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 the § Université de Bourgogne, Laboratoire
de Zoologie, Unité Mixte de Recherche 5548, CNRS,
"Développement, Communication Chimique," 6 Bd. Gabriel,
21000 Dijon, France
Received for publication, April 9, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Two novel antimicrobial peptides, which we
propose to name termicin and spinigerin, have been isolated from the
fungus-growing termite Pseudacanthotermes spiniger
(heterometabole insect, Isoptera). Termicin is a 36-amino acid residue
antifungal peptide, with six cysteines arranged in a disulfide array
similar to that of insect defensins. In contrast to most insect
defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria
and fungi. Termicin and spinigerin show no obvious sequence
similarities with other peptides. Termicin is constitutively present in
hemocyte granules and in salivary glands. The presence of termicin and
spinigerin in unchallenged termites contrasts with observations
in evolutionary recent insects or insects undergoing complete
metamorphosis, in which antimicrobial peptides are induced in the fat
body and released into the hemolymph after septic injury.
The termite species Pseudacanthotermes spiniger, like
the other members of the Macrotermitinae, depends for its nutrition on
a symbiotic fungus of the basidiomycete genus Termitomyces (1). This fungus grows inside the nests of the termites on piles of
fecal pellets; it predigests the lignocellulosic substances and is
responsible for food supply in cellulases. Macrotermitinae also
live in symbiosis with anaerobic bacteria present in their posterior
gut, which are in part responsible for the complete digestion of
cellulose. In this environment inhabited with microorganisms, how do termites protect themselves?
The first line of defense of insects against pathogens is the cuticle.
Once this barrier has been breached, their defense reactions rely both
on cellular and humoral mechanisms. The cellular aspects include
phagocytosis and encapsulation of invading microorganisms (for a
review, see Ref. 2). The humoral facet involves the activation of
proteolytic cascades leading to melanization and coagulation. In the
evolutionary recent insect orders, the best characterized aspect of the
humoral immune response is the rapid synthesis of antimicrobial
peptides/polypeptides by the fat body and certain blood cells and
release of these factors into the hemolymph after bacterial challenge.
Since the first report of an inducible antibacterial peptide from an
insect, cecropin from the moth Hyalophora cecropia (3), more
than 200 antimicrobial peptides/polypeptides have been characterized in
insects. On the basis of their structural features, the peptides are
classified into three classes: (i) linear peptides, devoid of cysteines
and forming In addition to the systemic antimicrobial response, insects frequently
show a local response, namely in barrier epithelia such as tracheal
epithelium, gut lining, and salivary glands (14). Termites frequently
lick their eggs and cover them with saliva, suggesting a protection of
the eggs against surrounding fungi and raising the question of the
presence of antifungal peptides in the saliva.
Hitherto, studies of the immune antimicrobial response had been focused
on insects belonging to recent groups or those that undergo complete
metamorphosis. This report presents the primary and secondary
structures of two antimicrobial peptides isolated from an insect with
an incomplete metamorphosis, the fungus-growing termite P. spiniger, from the order of Isoptera. The first molecule is a
novel cysteine-rich 36-residue peptide with a disulfide array identical
to that of insect defensins. This peptide is strongly antifungal and
only weakly affects several Gram-positive bacteria. In addition, three
isoforms of a smaller peptide, which is devoid of cysteines and
active against bacterial and fungal strains, were also characterized.
Furthermore, we show that (i) the termite antimicrobial peptides are
constitutively present and apparently not induced by bacterial
challenge as has been observed in all insect species so far
investigated and (ii) the cysteine-rich peptide is present in hemocyte
granules and in the salivary glands of P. spiniger.
Insects and Immunization
P. spiniger colonies were established at the
laboratory CNRS UMR 5548 "Développement Communication
Chimique" in Dijon from pairs of alates collected in Gabon in 1988. Females and males of the isopteran P. spiniger dealate
imagos received a 2-µl injection of a mixture of 2500 cells of
Micrococcus luteus (Gram-positive) and 2500 cells of
Escherichia coli 1106 (Gram-negative) obtained after
37 °C overnight cultures. After immunization, the termites were kept
at 25 °C in a moist chamber for 24 h and then frozen in liquid
nitrogen until used.
Acidic Extraction and Purification
Whole Body Extraction
Termites (200) were reduced to powder in a mortar containing
liquid nitrogen. The peptides were extracted at pH 3 in 100 ml of 0.1%
trifluoroacetic acid containing the protease inhibitor aprotinin
(10 µg/ml final concentration) and phenylthiourea (20 µM final concentration) as a melanization inhibitor.
Extractions were performed in an ice-cold water bath with gentle
shaking for 30 min. After centrifugation (14,000 × g
for 30 min at 6 °C), the supernatant was applied onto a Sep-Pak Vac
C18 cartridge (5 g of phase, WatersTM), and the peptides
were eluted with 5, 40, and 80% acetonitrile in 0.05% trifluoroacetic
acid. The 40% eluted fraction was lyophilized before being subjected
to purification.
HPLC Purification: Antifungal Molecule
Step 1--
The 40% Sep-Pak fraction was subjected to
reversed-phase (RP)1
chromatography on an Aquapore RP-300 C8 column (250 × 7 mm; Brownlee) equilibrated with 2% acetonitrile in 0.05%
trifluoroacetic acid. Separation of the fractions was performed with a
linear gradient of 2-60% acetonitrile in 0.05% trifluoroacetic acid
over 120 min at 1.3 ml/min.
Step 2--
The fractions exhibiting exclusively antifungal
activity were further purified by size exclusion chromatography using
serially linked HPLC columns (Ultraspherogel SEC 3000 and SEC 2000, 7.5 × 300 mm; Beckman). Elution was performed with 30%
acetonitrile in 0.05% trifluoroacetic acid at 0.4 ml/min.
Step 3--
The active peptide was purified on the same column
and at the same flow rate as in step 1 by using a biphasic gradient of acetonitrile in 0.05% trifluoroacetic acid from 2 to 15% over 10 min
and from 15 to 45% over 120 min.
Step 4--
The fourth step of purification was performed on an
analytical Aquapore OD-300 column (220 × 4.6 mm; Brownlee)
developed with a linear biphasic gradient of acetonitrile in acidified
water from 2 to 15% over 10 min and from 22 to 32% over 50 min with a
flow rate of 0.8 ml/min and at a temperature of 30 °C.
Last Step of Purification--
The last purification step was
carried out on a narrow bore C18 RP column (Delta Pak
HPIC18, 2 × 150 mm; Waters) at 30 °C with a flow
rate of 0.2 ml/min using a biphasic gradient of acetonitrile in 0.05%
trifluoroacetic acid from 2 to 17% over 10 min and from 17 to 27%
over 40 min.
HPLC Purification: Antimicrobial Peptide
Step 1--
Step 1 was identical to that described above
for antifungal molecule purification.
Step 2--
The second step of the purification run was
performed on an Aquapore OD-300 column as described above. Elution was
performed with a biphasic gradient of acetonitrile in 0.05%
trifluoroacetic acid from 2 to 18% over 10 min and from 22 to 42%
over 100 min at 0.8 ml/min.
Last Step--
The fraction containing the antimicrobial
molecule was finally purified to homogeneity using the narrow bore
column (see last step of purification of the antifungal molecule
above). The column was equilibrated in 0.05% trifluoroacetic acid and
developed with the same linear gradient of acetonitrile as described
above for step 2.
All purification steps were performed on a Beckman Gold HPLC system
equipped with a Beckman 168 photoarray detector or with an all-PEEK
Waters HPLC system (Waters model 626) attached to an absorbance
detector (Waters 486). In all HPLC purification steps, the column
effluent was monitored by absorbance at 225 nm.
Blood Cell/Salivary Gland Collection, Extraction, and Peptide
Purification
Blood Cells
Hemolymph from naive (700) and immune-challenged (3000) animals
was collected from the thorax and diluted in 10 volumes of an
anticoagulant buffer (carbonate at pH 6.8) supplemented with aprotinin.
The hemolymph was then centrifuged at 800 × g at
4 °C for 15 min to collect the blood cells.
Salivary Glands
Salivary glands were dissected from the thorax of naive (90) and
immunized (200) insects in a 10 mM phosphate-buffered
saline (PBS) at pH 7.4. After washing twice in PBS, the glands were
snap frozen in liquid nitrogen.
Peptides were extracted from the blood cells and the salivary glands by
sonication (3 × 20 s) at medium power in the presence of 2 M acetic acid in an ice-cold water bath. Extraction was
continued overnight in the 2 M acetic acid supplemented
with aprotinin. After centrifugation at 12,000 × g at
4 °C for 30 min, the different supernatants were applied onto
Sep-Pak light C18 cartridges, and the peptides were eluted
with 5 and 80% acetonitrile in 0.05% trifluoroacetic acid. After
lyophilization, the resuspended 80% Sep-Pak fractions were injected
onto a RP column (see last step of purification of the antifungal
peptide). Separations were performed at 30 °C with a linear gradient
of acetonitrile from 2 to 80% in 0.05% trifluoroacetic acid over 120 min at 0.2 ml/min.
Capillary Zone Electrophoresis (CZE)
Peptide purity was ascertained on 1 nl of fractions using a
270A-HT electrophoresis system (Applied Biosystems, Inc.). Analysis was
performed in the conditions described by Lamberty and colleagues (13).
Mass Measurement by MALDI-TOF-MS
Mass analysis of the samples was performed on a Bruker (Bremen)
BIFLEX IIITM matrix-assisted laser desorption/ionization
time of flight mass spectrometer as described previously (15).
Microsequence Analysis
Automated Edman degradation of the purified peptides and
detection of the phenylthiohydantoin derivatives were carried out using
a pulse liquid automatic sequenator (Applied Biosystems Inc.,
model 473A).
Reduction and S-Pyridylethylation
Purified peptide (1 nmol) was subjected to reduction with
dithiothreitol and alkylation with 4-vinylpyridine in the presence of
guanidinium hydrochloride using a procedure already described (16).
Bioassays
The microbial strains used to determine antimicrobial activity
were those already reported in previous studies (10, 17). During the
purification procedure, antimicrobial activity was monitored by liquid
growth inhibition assays against M. luteus, E. coli 363, and Neurospora crassa as described previously
(18). The minimal inhibitory concentrations (MICs) of the synthetic and
recombinant peptides were determined against various fungal and
bacterial strains using the same procedure.
Under conditions where the antifungal assay was performed on hyphae,
spores were allowed to pregerminate in 1:2 potato dextrose broth
(Difco) for 24 h at 30 °C. Peptide solutions were then added to
the appropriate final concentration (2-fold serial dilutions from 100 to 0.2 µM), and incubation was continued for a further 24 h. When the antifungal assay was carried out in the presence of
salt, the 1:2 potato dextrose broth medium was prepared in PBS,
supplemented with either 1 mM CaCl2 or 50 mM KCl.
Peptide Synthesis
Peptide synthesis was performed by classical Fmoc methodology as
described previously (12). After deprotection, the peptide was
subjected to solid-phase extraction onto a Sep-Pak Vac C18 cartridge (10 g of phase; Waters) and eluted with 50% acetonitrile in
0.05% trifluoroacetic acid, concentrated, and purified to homogeneity on RP column (Aquapore C18, 250 × 10 mm; Brownlee).
Elution was performed at 2.5 ml/min with a linear biphasic gradient of
acetonitrile from 2 to 55% over 60 min.
Production of Recombinant Antifungal Peptide and Purification
Strains, Media, and Plasmids
The strains used for cloning and expression of recombinant
peptide have already been described (13). The following
oligonucleotides were used for construction of the synthetic gene: OJL
256 (5'-AGCTTGGATAAAAGAGCTTGTAATTTCCAATCTTGTTGGGCCACGTGT-3'), OJL 257 (5'-CAAGCTCAACATTCTATTTACTTTAGAAGAGCTTTCTGT-3'), OJL 258 (5'-GATAGATCTCAATGTAAATGTGTTTTTGTTAGAGGTTAAG-3'), OJL 261 (5'-TCGACTTAACCTCTAACAAAAACACATTTACATTGAGATCTATCACAGAAAGC-3'), OJL 262 (5'-TCTTCTAAAGTAAATAGAATGTTGAGCTTGACACGTGGC-3'), and OJL 263 (5'-CCAACAAGATTGGAAATTACAAGCTCTTTTATCCA-3').
The plasmid containing the synthetic gene and the yeast shuttle vector
were constructed as described previously (11, 13), and insertion
success was assessed by sequencing. Yeast cells were transformed using
the lithium acetate method (19).
Purification of Recombinant Peptide
The recombinant peptide was purified from the yeast culture
supernatant using the procedure described previously (13). Elution was
performed at 2.5 ml/min with a gradient of acetonitrile from 2 to 17%
over 10 min and from 17 to 27% over 60 min. Finally, the fraction
containing the peptide was lyophilized and kept as dry powder.
Enzymatic Digestions
Arginyl Endopeptidase Treatment
The S-pyridylethylated peptide (600 pmol) was treated
with arginyl endopeptidase according to the procedure recommended by the manufacturer (Takara, Otsu, Japan) at an enzyme/substrate ratio of
1:50 (w/w) for 16 h at 37 °C. Peptidic fragments were separated
on a Delta Pak HPI C18 column (2 × 150 mm; Waters)
with a 0-60% linear gradient of acetonitrile in 0.05%
trifluoroacetic acid over 90 min at 0.2 ml/min, at 30 °C.
Thermolysin Digestion
Recombinant and native peptides were treated with thermolysin
from Bacillus thermoproteolyticus (Roche Molecular
Biochemicals), and the fragments of digestion were analyzed in
conditions previously reported (13).
Immunohistochemistry
Recombinant antifungal peptide (termicin) was used to raise
rabbit polyclonal antibodies. The serum was purified using
immunoaffinity. Blood films from naive and immunochallenged dealate
imagos were fixed on polylysine coated slides by immersion in 4%
formaldehyde, 0.5% glutaraldehyde in 0.1 M phosphate
buffer, pH 7.4. The slides were rinsed, dehydrated, and stored at
Isolation and Identification of Antimicrobial Peptides from P. spiniger
To isolate and characterize molecules responsible for
antimicrobial activity in the termite P. spiniger
(Isoptera), 200 dealate imagos were challenged by injection of a
mixture of M. luteus and E. coli 1106 to induce
an immune response. After 24 h, peptides were extracted in acidic
conditions. The extract was prepurified by solid-phase extraction on a
RP cartridge and eluted with various solutions of acetonitrile
in 0.05% trifluoroacetic acid (see "Experimental Procedures").
Here we have focused on the analysis and purification of the 40%
Sep-Pak fraction. This fraction was subjected to RP chromatography.
Each individually collected peak was lyophilized and reconstituted in
MilliQ water. The presence of antimicrobial activity was monitored by
liquid growth inhibition assays on three test microorganisms selected
arbitrarily for their high sensitivity to insect antimicrobial peptides
(18) without regard for the pathogenicity to the termite: the
Gram-positive strain M. luteus, the Gram-negative strain
E. coli 363, and the filamentous fungus N. crassa
(Fig. 1). Four fractions were exclusively
active against N. crassa. Eight fractions showed activity
against N. crassa and M. luteus. Finally, five
fractions, in particular fraction F1 (Fig. 1), were found to be active
against the three species tested. In a control experiment with
unchallenged animals, similar levels and diversity of the antimicrobial
activity were observed (data not shown).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices (the prototype of this family are the insect cecropins (4)), (ii) peptides with an overrepresentation in proline
and/or glycine residues, and (iii) open-ended cyclic peptides containing cysteine residues (5). Cecropins, proline-rich, and
glycine-rich peptides are essentially active against Gram-negative cells, but their activity spectrum sometimes includes Gram-positive bacteria as targets (6). Two recent reports have also indicated that
cecropins can also exert antifungal properties (7, 8). Among the open
ended cysteine-rich peptides, insect defensins are the most widespread
(9). Insect defensins are mainly active against Gram-positive bacteria,
but some activity is occasionally recorded against Gram-negative cells
and fungi (6). All insect defensins harbor a consensus motif of six
cysteine residues involved in the formation of three disulfide bridges.
In addition to defensins, three cysteine-containing antifungal peptides
have been isolated from insects: (i) drosomycin, an antifungal peptide
isolated from the fruit fly Drosophila melanogaster, which
consists of 44 amino acids and has eight cysteine residues (10, 11);
(ii) thanatin, a 2-kDa peptide carrying two cysteine residues isolated
from the hemipteran Podisus maculiventris (12), which has
both bactericidal and fungicidal properties; and (iii) heliomicin, a
44-residue peptide recently isolated from immune-induced larvae of the
lepidopteran Heliothis virescens (13) with the same cysteine
arrangement as that present in insect defensins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C in air-tight boxes until required. Staining was performed
essentially following a procedure already described (20). Cells were
incubated overnight at 4 °C in an affinity-purified rabbit
anti-termicin polyserum diluted to 1:2000 in PBS containing 0.1% Tween
20. The secondary antibody used was horseradish peroxidase-linked goat
anti-rabbit IgG (Sigma).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reversed-phase HPLC separation of the acidic
extract obtained from immune-activated adults of P. spiniger. After prepurification by solid-phase
extraction, the 40% elution fraction 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 activity against M. luteus,
(hatched rectangles), E. coli 363 (black rectangles), and N. crassa
(white rectangles) was measured by liquid growth
inhibition assays. Insets show the spectra obtained by
MALDI-TOF-MS of the antifungal molecule (A) present in the
zone marked by a line and the antimicrobial peptides
contained in fraction F1 (B) characterized in this study
after the last step of purification. The active compound contained in
fraction F2 was further purified to homogeneity and sequenced (see
"Results").
The fractions exclusively active against N. crassa were pooled and applied to size exclusion chromatography (data not shown). Aliquots of the collected fractions were tested against N. crassa. The most active fraction was further purified by a three-step RP HPLC procedure (see "Experimental Procedures"). Finally, the antifungal peptide was purified to homogeneity, as judged by CZE (data not shown). Mass measurement by MALDI-TOF recorded a single peak at 4216.05 MH+ (Fig. 1, inset A). Among the fractions active on the three strains tested, fraction F1, exhibiting the strongest activity against M. luteus, was subjected to two additional RP chromatographies. After the last step of purification, CZE showed the presence of two peaks (data not shown). The presence of two peaks at 3001.7 and 2650.5 MH+ recorded by MALDI-TOF-MS confirmed this heterogeneity (Fig. 1, inset B).
Structure Determination of the Antimicrobial Peptides
A Novel Cysteine-rich Antifungal Peptide--
The native
antifungal peptide (1 nmol) at 4216.05 MH+ was subjected to
reduction and alkylation with 4-vinylpyridine. The reaction product was
analyzed both by mass spectrometry (MALDI-TOF) and Edman degradation.
The mass of the pyridylethylated peptide (4852.2 MH+)
exceeded that of the native peptide by 636.15 Da, which corresponds to
the mass of six pyridylethylated groups; this suggests the presence
of six cysteine residues. Sequencing of the pyridylethylated peptide
yielded a sequence of 34 residues:
AC*NFQSC*WATC*QAQHSIYFRRAFC*DRSQC*KC*VFV, where C* stands
for a pyridylethylated cysteine. The calculated molecular mass of the
N-terminal peptidic portion (4639.5 MH+) is lower by 212.7 Da than the measured mass of the native molecule at 4852.2 MH+, indicating that it does not represent the full
sequence. To gain information on the C-terminal part of the molecule,
the pyridylethylated peptide was subjected to digestion by
endoproteinase Arg-C. Peptidic fragments were purified by RP HPLC (data
not shown) and analyzed by Edman degradation. One of the peptidic
fragments gave the sequence C*VFVR (molecular mass measured at 728.9 MH+). This sequence indicated that the pyridylethylated
molecule was cleaved after Lys30 and Arg35 (see
Fig. 2C for numbering). The
mass difference of 55.5 Da between the mass recorded by MALDI-TOF for
the pyridylethylated peptide (4852.2 MH+) and the
calculated mass of the peptide (4796.7 MH+), including the
C-terminal sequence, suggested that the last residue corresponds to a
glycine residue (57 Da). The additional mass difference of 1.5 Da
further suggested that the peptide carries an amidation at its C
terminus. The full-length sequence of the antifungal compound is
presented in Fig. 2C. Data bank analysis (BLAST program in
Swiss-Prot; Ref. 21) did not show obvious sequence similarities of this
peptide with other known peptides or proteins. This novel 36-residue
cysteine-rich antifungal peptide from the termite P. spiniger was named termicin.
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Production of Recombinant Termicin
A heterologous expression system was used to produce sufficient
amounts of termicin to determine its disulfide array and its activity
spectrum. Cassette assembly, cloning procedures, and yeast
transformation techniques were as described earlier (11, 13). Termicin
was purified from the yeast culture supernatant by two steps of
solid-phase extractions (RP and cation exchange chromatography) and a
preparative RP chromatography. The identity and purity of the
recombinant peptide was controlled by CZE analysis and MALDI-TOF-MS
measurement. The mass spectrometry value at 4216.9 MH+,
observed for the recombinant peptide, exceeded the mass of the native
molecule (4216.05 MH+) by 0.85 Da, suggesting a C-terminal
amidation of the native peptide (data not shown). This hypothesis was
confirmed using CZE analysis of coinjected native and yeast-expressed
peptide. A control experiment was carried out by coinjecting amidated
and nonamidated Anopheles gambiae cecropin (data not shown).
In both cases, the same difference in retention times was observed
between the nonamidated and amidated peptide forms, suggesting that
native termicin has a glycine -amide as C terminus. The identity of recombinant termicin to the natural compound was established (i) by
coelution studies by RP HPLC and (ii) by determination of the MIC
against N. crassa. Both studies confirmed the identity of the recombinant termicin.
Determination of the Disulfide Array of Termicin
The calculated mass of the antifungal peptide obtained after Edman
degradation (4221.9 MH+) was 5.85 Da in excess of that
measured by MALDI-TOF-MS for the native molecule (4216.05 MH+). This indicated that the six cysteines were engaged in
the formation of three disulfide bonds. Analysis of the primary
structure of the antifungal peptide showed that it harbors the
cysteine-stabilized motif described in insect defensins (22). In
most cases, the position of the disulfide bridges has been determined
and indicates identical pairing of Cys(1)-Cys(4), Cys(2)-Cys(5),
and Cys(3)-Cys(6) (numbers in parenthesis relative to the cysteine order). Since not enough native peptide was available, the disulfide array was determined by thermolysin treatment on recombinant peptide assuming that recombinant and native peptides only differed by the
C-terminal amidation in the native molecule. The peptidic fragments
resulting from the thermolysin digest were separated by RP HPLC, and
the molecular masses of the major peaks were determined by
MALDI-TOF-MS. Among the different fractions analyzed by MS, two major
fractions (fractions I and II) were found to be pure enough for
sequencing by Edman degradation (Fig. 2A). The different phenylthiohydantoin-derivative signals obtained in each cycle for
fraction I were in agreement with the connection of the fragment Ala22-Cys31 (upper numbering relative to the
residue number in the primary sequence of the termicin) (i) to fragment
Ala1-Asn3 through a disulfide bridge between
Cys24 and Cys2 and (ii) to the peptidic portion
Phe4-Ser16 through two intramolecular bonds
(Cys29-Cys7 and
Cys31-Cys11) (Fig. 2B). Regarding
the sequencing of fraction II, an additional signal of
phenylthiohydantoin-Trp in cycle 1 compared with the data obtained for
fraction I established that thermolysin had cleaved the linkage between
Cys7 and Trp8 (Fig. 2B). The
difference of 17.7 Da observed between fraction I and fraction II was
the consequence of this cleavage, resulting in the addition of a
molecule of water. The calculated masses obtained for both peptidic
fragments (2958.3 and 2976.3 MH+, respectively) were in
agreement with the molecular masses measured by MALDI-TOF. In
conclusion, the results were compatible with the presence of the
conserved insect defensin disulfide array: Cys2(1)-Cys24(4),
Cys7(2)-Cys29(5) and
Cys11(3)-Cys31(6) (Fig. 2C). To
assess whether the cysteine array of native termicin was identical to
that stet for the recombinant molecule, native termicin was
subjected to thermolysin digestion. The digestion products were
directly analyzed by MALDI-TOF-MS (data not shown). Observation of
identical molecular masses to that measured for fractions I and II (see
above and Fig. 2) confirmed that the cysteine scaffolds of native and
recombinant termicins were identical.
A Novel Linear Antimicrobial Molecule
Although the antimicrobial compounds contained in fraction F1
could not be separated by the third step of HPLC purification (see
above), the fraction was submitted to Edman degradation. Two
phenylthiohydantoin-derivative signals were obtained at each sequencing
cycle. However, from the fourth cycle onwards, it appeared that one of
the sequences corresponded to a truncated form of the other. The masses
of both peptides calculated from the two primary structures (2650.4 and
3001.8 MH+, respectively) were in total agreement with the
masses measured after MALDI-TOF MS (2650.5 and 3001.7 MH+,
respectively). The data obtained by Edman degradation showed that the
peptide at 2650.5 MH+ corresponded to the molecule at
3001.7 MH+ lacking the first three amino acids
(His-Val-Asp). Another peptide isoform (2517.7 MH+) present
in a neighboring fraction (F2, see Fig. 1) was purified, and the full
sequence was obtained by Edman degradation. It corresponded to the
3001.7 MH+ peptide lacking the last four residues
(Leu-Thr-Arg-Leu). The sequences obtained for the three peptide
isoforms are shown in Fig. 3. The peptide
exhibiting the longest primary sequence was synthesized by the Fmoc
method to further investigate its biological properties. This
25-residue linear antimicrobial peptide was named spinigerin from the
species spiniger.
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Antimicrobial Activity Spectrum of the Two P. spiniger Antimicrobial Peptides
Activity Spectrum of Termicin-- Pure recombinant peptide was tested on various filamentous fungi, yeast strains, and Gram-positive and -negative bacteria. Termicin had marked activity against the spores of Nectria hematococca, N. crassa, Fusarium culmorum, and F. oxysporum (Table I). It was less active against Trichoderma viride, and inactive against the spores of Aspergillus fumigatus and Beauveria bassiana at the highest concentration tested (100 µM). However, morphological alterations of A. fumigatus hyphae were observed (Fig. 4). Termicin had a similar level of activity against three out of the four yeast strains tested (Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae) but was inactive against Candida glabrata at the highest concentration tested (100 µM). Termicin showed weak activity against some Gram-positive bacteria (e.g. Streptococcus pyogenes, Bacillus megaterium, and M. luteus), but was inactive against Aerococcus viridans and Staphylococcus aureus. No activity against Gram-negative bacteria was detected even at 100 µM.
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Similar antifungal properties against N. crassa (MIC = 0.2-0.4 µM) were observed when activity was tested in the presence of 50 mM KCl or at a physiological ionic strength (137 mM NaCl). When the medium was supplemented with 1 mM CaCl2, a 2-fold loss in activity was recorded against N. crassa (MIC = 0.4-0.8 µM). A kinetic study of termicin action against N. crassa revealed that a 3-h incubation was sufficient to kill all of the spores at a concentration of 3 µM (10-fold higher than its MIC value).
Activity Spectrum of Spinigerin-- After purification, the integrity and purity of synthetic spinigerin were assessed by CZE and MALDI-TOF-MS.
Among the six Gram-positive bacteria tested, only M. luteus and B. megaterium were affected by the peptide (Table II). The peptide showed no activity against B. subtilis, B. thuringiensis, S. pyogenes, or S. aureus. The growth of the Gram-negative strains Enterobacter cloacae and Erwinia carotovora was not affected by 100 µM of spinigerin. However, the peptide was active against E. coli SBS363, E. coli D22, and Salmonella typhimurium while being less active against Klebsiella pneumoniae and Pseudomonas aeruginosa. Spinigerin was active against most of the fungal strains tested (e.g. N. hematococca, T. viride, F. culmorum, and N. crassa) (Table II) but showed no activity against the fungus B. bassiana even at 100 µM. The peptide also showed activity against the yeast strain C. albicans.
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Additional assays on N. crassa were performed in 1:2 potato dextrose broth medium supplemented with PBS (137 mM NaCl), 1 mM CaCl2, or 50 mM KCl. Spinigerin activity was not affected by the presence of PBS. The addition of 1 mM CaCl2 or 50 mM KCl induced a 2-fold decrease in spinigerin activity against N. crassa (MIC = 6-12 µM versus 3-6 µM).
Tissue Localization of Termicin
Blood cell and salivary gland extracts were prepared from naive
and immune-challenged animals. The extracts were directly applied onto
Sep-Pak C18 cartridges. The 80% Sep-Pak elutions were
further fractionated by RP HPLC. Since the amount of peptide available
was low, no antifungal assay was performed. The presence of termicin
was directly determined by MALDI-TOF-MS on fractions eluting with the
retention time previously established for termicin (see Fig. 1). One
peak in each of the four extracts contained a molecular mass at 4216.0 MH+. The four fractions were subjected to a 10-residue
N-terminal sequencing by Edman degradation to ascertain the identity of
the molecule at 4216.0 MH+. Indeed, the N-terminal
sequencing confirmed the identity of the molecule as termicin, and the
mass spectrometry analysis confirmed its integrity. These data
indicated that termicin was produced or stored in the salivary glands
and the hemocytes of both naive and immune-activated adults (data not
shown). Furthermore, as illustrated in Fig.
5, termicin-immunoreactive material was
present in the granules of large blood cells.
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DISCUSSION |
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This paper describes the isolation and characterization of the first two antimicrobial peptides from an isopteran insect, the termite P. spiniger. These are termicin, a peptide exhibiting essentially antifungal properties, and spinigerin, a molecule with antifungal and antibacterial activities.
Termicin, in addition to having six cysteine residues, shares the
disulfide array of the insect defensins, including the
cysteine-stabilized motif (22). This leads to the proposal that
termicin has a similar
three-dimensional arrangement to that
of insect defensins. However, with the exception of the six cysteine
residues, termicin shows little sequence homology with insect
defensins. The closest sequence similarity observed is with sapecin B
from Sarcophaga peregrina (23) (Fig.
6), while the activity of termicin is
closer to the antifungal peptides such as plant defensins (24), drosomycin from D. melanogaster (10), and heliomicin from
the lepidopteran H. virescens (13). Whereas plant defensins
and drosomycin have four disulfide bridges, termicin and heliomicin have only three disulfide bridges.
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Recombinant termicin showed marked activity against most of the fungal species tested, with MICs generally higher than those observed for the Heliothis antifungal reference peptide, heliomicin (see Table I). Interestingly, the peptide showed no obvious activity against the spores of the fungus A. fumigatus as observed by spectroscopy analysis. However, observation of the resulting hyphae by light microscopy revealed morphological distortions (Fig. 4B). When the antifungal assay was performed directly on hyphae of A. fumigatus, microscopic observations revealed that termicin is able to perforate the hyphal wall. Occasionally, local leakage of cytosolic material was observed (see Fig. 4C). In this case, termicin behaves like a "morphogenic" defensin, according to the definition proposed for plant defensins, causing reduced hyphal elongation with increased branching (25). Termicin exhibited some activity against the yeast strains C. albicans and C. neoformans at a 2-fold higher concentration than heliomicin. Surprisingly, recombinant termicin is active against the yeast strain S. cerevisiae (MIC = 6-12 µM) in which it was expressed (Table I). The average recovery of peptide with this expression system was of 1.5 mg/liter of culture medium (0.3 µM), and termicin had no effect on yeast growth. In contrast to heliomicin, termicin exhibited antibacterial activity against several Gram-positive strains. Termicin kills the Gram-positive bacteria at MICs significantly higher than those of insect defensins (e.g. Phormia defensin) used as controls (see Table I). Unlike Phormia defensin, termicin was inactive against Gram-negative bacteria even at the highest concentration tested (100 µM) (see Table I).
In addition to termicin, termites produce a linear 25-amino acid
antimicrobial peptide named spinigerin, with both antifungal and
antibacterial properties. Two truncated (N- and C-terminally) spinigerin isoforms (22 and 21 residues, respectively) were isolated and entirely purified (see Fig. 3). One hypothesis for the presence of
the two shortest forms of the 25-amino acid peptide is degradation of
this linear molecule by proteases present either in the hemolymph or
released during extraction from whole termites. However, the possibility cannot be exclude that the shorter forms may be modified enzymatically to generate forms with differential target cell specificities. Data bank analysis of spinigerin did not show sequence similarities with other known antimicrobial peptides. However, the
absence of cysteines and the distribution of hydrophobic and polar
amino acids in the primary structure indicate that the peptide may
adopt a -helical structure like that of insect cecropins. This
prompts a comparison between the biological properties of spinigerin
and two representatives of the amphipathic
helical antimicrobial
peptides: Aedes aegypti cecropin (8) and PGLa from
Xenopus laevis (26) (see Table II). Spinigerin showed
activity against two of the six Gram-positive bacteria tested, namely
M. luteus and B. megaterium. It was also found to
be active against most of the Gram-negative bacteria tested, although
the MIC values observed were higher than those obtained for the
Gram-positive cells. Aedes cecropin and PGLa were more
effective than spinigerin against most of the bacteria tested (Table
II). Spinigerin, when assayed against filamentous fungi, showed
activity equivalent to that of Aedes cecropin and PGLa.
Against the yeast strain C. albicans, spinigerin was more
efficient than the two
-helical peptides used as references.
The data presented here establish that a member of the insect order of
Isoptera produces a set of antimicrobial peptides in response to a
septic injury. Antimicrobial peptides are key components of innate
immunity against bacteria and fungi in invertebrates, vertebrates, and
plants. In insects, septic injury leads to rapid synthesis of these
peptides by the fat body and certain blood cells, followed by their
release into the hemolymph. However, in our study on the isopteran
P. spiniger, the antimicrobial molecules appear to be
constitutively present. A control experiment processed with
unchallenged animals gave no indication that injection of bacteria
modified the spectrum and/or the level of the antimicrobial compounds.
Furthermore, no antimicrobial activity had been detected in the
hemolymph collected from naive and immune-challenged animals. In fact,
the antifungal peptide termicin was detected in hemocyte granules and
in salivary glands prepared from unchallenged and challenged insects.
The presence of mature peptide in salivary glands suggests a protective
role for the molecule against fungi, since termites smear their eggs
with saliva during egg development. The results presented here contrast
with those so far reported for other insects, where bacterial challenge
induces the production of antimicrobial peptides and their subsequent
release into the hemolymph. However, storage of antimicrobial peptides
in granular cells has already been reported in other organisms
including humans. For example, antimicrobial peptides are
constitutively present in hemocytes of the shrimp Penaeus
vannamei (27), the horseshoe crab Tachypleus
tridentatus (28), and the mussel Mytilus
galloprovincialis (29). In T. tridentatus, microbial
stimulation induces degranulation and release into the extracellular
fluid of different immune defense substances such as tachyplesins (28),
big defensin (30), and tachycitin (31). In the mussel M. galloprovincialis, bacterial challenge triggers the release of the
antimicrobial peptide MGD1 from the hemocytes into the hemolymph (32).
A similar mechanism may occur in P. spiniger. In essence,
therefore, two modes of fighting infections by antimicrobial peptides
appear to exist in insects: (i) transcription of the genes coding for
antimicrobial peptides, mainly in the fat body, after septic injury and
rapid release into the hemolymph of the antimicrobial compounds and (ii) constitutive production and storage of the antimicrobial substances, particularly in hemocytes, and release of the peptides into
the blood after immune challenge.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. J. P. Briand and J. P. Roussel for spinigerin synthesis. We are grateful to C. Hetru for help and discussions and to M. Schneider for technical assistance. We thank L. Ehret-Sabatier and N. Cavusoglu for performing the mass spectrometry. We thank Dr. Philaberg Irving for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by CNRS, the University Louis Pasteur of Strasbourg, Rhône-Poulenc Agro, and the Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The protein sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P82321 (for termicin) and P82357 (for spinigerin).
¶ To whom correspondence should be addressed. Tel.: 33388417062; Fax: 33388606922; E-mail: P.Bulet@ibmc.u-strasbg.fr.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M002998200
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ABBREVIATIONS |
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The abbreviations used are: RP, reversed-phase; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; MS, mass spectrometry; MIC, minimal inhibitory concentration; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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