Insect Immunity

Mireille Lamberty, Daniel Zachary, René Lanot, Christian Bordereau, Alain

Robert, Jules Hoffmann, Philippe Bulet

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To cite this version:

Mireille Lamberty, Daniel Zachary, René Lanot, Christian Bordereau, Alain Robert, et al.. Insect Immunity. Journal of Biological Chemistry, 2001, 276 (6), pp.4085-4092. 10.1074/jbc.M002998200. hal-03828761

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# Insect Immunity

## CONSTITUTIVE EXPRESSION OF A CYSTEINE-RICH ANTIFUNGAL AND A LINEAR ANTIBACTERIAL PEPTIDE IN A TERMITE INSECT*

Received for publication, April 9, 2000, and in revised form, October 25, 2000
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M002998200

Mireille Lamberty‡, Daniel Zachary‡, René Lanot‡, Christian Bordereau§, Alain Robert§, Jules A. Hoffmann‡, and Philippe Bulet¶¶

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

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 α-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.

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

<footnote>* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>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).</footnote>

<footnote>¶ To whom correspondence should be addressed. Tel.: 33388417062; Fax: 33388606922; E-mail: P.Bulet@ibmc.u-strasbg.fr.</footnote>
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*.

## EXPERIMENTAL PROCEDURES

### 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, Waters™), 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)<fnref n="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 III™ 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.

<footnote n="1">¹ 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.</footnote>
## 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'-AGCTTGGA-TAAAAGAGCTTGTAATTTCCAATCTTGTTGGGCCACGTGT-3'), OJL 257 (5'-CAAGCTCAACATTCTATTTACTTTAGAAGAGCTTTCTGT-3'), OJL 258 (5'-GATAGATCTCAATGTAAATGTGTTTTTGTTAGAGG-TTAAG-3'), OJL 261 (5'-TCGACTTAACCTCTAACAAAAACACATTT-ACATTGAGATCTATCACAGAAAGC-3'), OJL 262 (5'-TCTTCTAAAG-TAAATAGAATGTTGAGCTTGACACGTGGC-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 -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

## 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% trifluoro-

<figure><img src="image_3.png" /><figcaption>FIG. 1. Reversed-phase HPLC separation of the acidic extract obtained from immune-activated adults of *P. spiniger*. After pre-purification by solid-phase extraction, the 40% elution fraction was analyzed on an Aquapore RP-300 C~8~ 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").</figcaption></figure>

acetic 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).

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 antimicrobial peptide (1 nmol) at 4216.05 MH⁺ was subjected to reduction and alkylation with 4-vinylpyridine. The reaction
<figure><img src="image_4.png" /><figcaption>FIG. 2. Determination of the disulfide array of the antifungal peptide termicin. Following thermolysin digestion, purification of the digest products by HPLC, and mass spectrometry measurements, two fractions (fractions I and II in A) were sequenced by Edman degradation. The sequences obtained for fractions I and II, including the connection of the cysteine residues, are reported in B. The disulfide array of the peptide is presented in C, where the *asterisk* represents the amidation of the glycine residue in the native molecule.</figcaption></figure>

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\*QAQHSIYFRRA-FC\*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.

## 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 α-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 αβ 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 set 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
<table><thead><tr><th>Fraction number</th><th>Calculated/measured mass (MH<sup>+</sup>)</th><th>Primary structure</th></tr></thead><tbody><tr><td>F1</td><td>3001.8/3001.7<br>2650.4/2650.5</td><td>HVDKKVADKVLLLLKQLRIMRLLTRL<br>KKVADKVLLLLKQLRIMRLLTRL</td></tr><tr><td>F2</td><td>2518.2/2517.7</td><td>HVDKKVADKVLLLLKQLRIMRL</td></tr></tbody></table>

**FIG. 3.** **Primary structure and mass measurements of the three isoforms of the antimicrobial peptide spinigerin.** The calculated molecular mass of the three isoforms was in total agreement with the molecular mass measured by MALDI-TOF mass spectrometry. Molecular masses are expressed in MH⁺, and the amino acid sequence is shown with one-letter amino acid codes.

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.

### 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.

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).

<caption>TABLE I</caption>

*Antimicrobial activity spectrum of recombinant termicin compared with that of heliomicin from H. virescens and Phormia defensin*

The minimal inhibitory concentration is expressed as final concentration in µM. Activity not detected at the highest concentration tested (100 µM) is indicated in the table by ND.

<table><thead><tr><th rowspan="2">Microorganisms</th><th colspan="3">Minimal inhibitory concentration</th></tr><tr><th>Termicin (P. spiniger)</th><th>Heliomicin (H. virescens)</th><th>Defensin (P. terranovae)</th></tr><tr><td></td><td colspan="3" style="text-align: center;">µM</td></tr></thead><tbody><tr><td colspan="4"><strong>Gram-positive bacteria</strong></td></tr><tr><td>A. viridans</td><td>ND</td><td>ND</td><td>0.05–0.1</td></tr><tr><td>B. megaterium</td><td>50–100</td><td>ND</td><td>0.05–0.1</td></tr><tr><td>M. luteus</td><td>50–100</td><td>ND</td><td>0.4–0.8</td></tr><tr><td>S. aureus</td><td>ND</td><td>ND</td><td>1.5–3</td></tr><tr><td>S. pyogenes</td><td>25–50</td><td>ND</td><td>1.5–3</td></tr><tr><td colspan="4"><strong>Gram-negative bacteria</strong></td></tr><tr><td>E. coli D22</td><td>ND</td><td>ND</td><td>1.5–3</td></tr><tr><td>E. coli SBS363</td><td>ND</td><td>ND</td><td>25–50</td></tr><tr><td>P. aeruginosa</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td colspan="4"><strong>Filamentous fungi</strong></td></tr><tr><td>A. fumigatus</td><td>ND</td><td>6–12</td><td>ND</td></tr><tr><td>B. bassiana</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>F. culmorum</td><td>0.2–0.4</td><td>0.2–0.4</td><td>1.5–3</td></tr><tr><td>F. oxysporum</td><td>0.8–1.5</td><td>1.5–3</td><td>3–6</td></tr><tr><td>N. crassa</td><td>0.2–0.4</td><td>0.1–0.2</td><td>3–6</td></tr><tr><td>N. haematococca</td><td>0.05–0.1</td><td>0.4–0.8</td><td>0.8–1.5</td></tr><tr><td>T. viride</td><td>6–12</td><td>1.5–3</td><td>6–12</td></tr><tr><td colspan="4"><strong>Yeast</strong></td></tr><tr><td>C. albicans</td><td>6–12</td><td>2.5–5</td><td>ND</td></tr><tr><td>C. glabrata</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>C. neoformans</td><td>6–12</td><td>2.5–5</td><td>ND</td></tr><tr><td>S. cerevisiae</td><td>6–12</td><td>ND</td><td>ND</td></tr></tbody></table>

**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*.

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
<figure><img src="image_5.png" /><figcaption>TABLE II
Antimicrobial activity spectrum of synthetic spinigerin compared with that of A. aegypti cecropin and PGLa from X. laevis</figcaption></figure>

The minimal inhibitory concentration is expressed as final concentration in µM. Activity not detected at the highest concentration tested (100 µM) is indicated in the table by ND.

<table><thead><tr><th rowspan="2">Microorganisms</th><th colspan="3">Minimal inhibitory concentration</th></tr><tr><th>Spinigerin<br>(P. spiniger)</th><th>Cecropin<br>(A. aegypti)</th><th>PGLa<br>(X. laevis)</th></tr><tr><td></td><td colspan="3" style="text-align: center;">µM</td></tr></thead><tbody><tr><td colspan="4"><strong>Gram-positive bacteria</strong></td></tr><tr><td><em>B. subtilis</em></td><td>ND</td><td>ND</td><td>0.5-1</td></tr><tr><td><em>B. thuringiensis</em></td><td>ND</td><td>ND</td><td>5-1</td></tr><tr><td><em>B. megaterium</em></td><td>0.8-1.5</td><td>10-25</td><td>0.5-1</td></tr><tr><td><em>M. luteus</em></td><td>0.8-1.5</td><td>10-25</td><td>0.5-1</td></tr><tr><td><em>S. pyogenes</em></td><td>ND</td><td>10-25</td><td>1-2.5</td></tr><tr><td><em>S. aureus</em></td><td>ND</td><td>ND</td><td>1-2.5</td></tr><tr><td colspan="4"><strong>Gram-negative bacteria</strong></td></tr><tr><td><em>E. cloacae</em></td><td>ND</td><td>50-100</td><td>ND</td></tr><tr><td><em>E. carotovora</em></td><td>ND</td><td>0.25-0.5</td><td>1-2.5</td></tr><tr><td><em>E. coli</em> SBS363</td><td>3-6</td><td>0.1-0.25</td><td>1-2.5</td></tr><tr><td><em>E. coli</em> D22</td><td>3-6</td><td>0.25-0.5</td><td>1-2.5</td></tr><tr><td><em>K. pneumoniae</em></td><td>50-100</td><td>1-2.5</td><td>10-20</td></tr><tr><td><em>S. typhimurium</em></td><td>3-6</td><td>1-2.5</td><td>10-20</td></tr><tr><td><em>P. aeruginosa</em></td><td>50-100</td><td>10-25</td><td>20-40</td></tr><tr><td colspan="4"><strong>Filamentous fungi</strong></td></tr><tr><td><em>B. bassiana</em></td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td><em>T. viride</em></td><td>1.5-3</td><td></td><td></td></tr><tr><td><em>N. crassa</em></td><td>3-6</td><td>6-12</td><td>2.5-5</td></tr><tr><td><em>F. culmorum</em></td><td>1.5-3</td><td>1-2.5</td><td>0.5-1</td></tr><tr><td><em>N. haematococca</em></td><td>0.4-0.8</td><td></td><td></td></tr><tr><td colspan="4"><strong>Yeast</strong></td></tr><tr><td><em>C. albicans</em></td><td>3-6</td><td>25-50</td><td>1-2.5</td></tr></tbody></table>

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.

DISCUSSION

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 αβ motif (22). This leads to the proposal that termicin has a similar αββ 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.

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
<figure><img src="image_6.png" /><figcaption>**FIG. 5.** Light photomicrograph of hemolymph smears of *P. spiniger* dealate imagos. The smears were incubated with an immunoaffinity-purified rabbit anti-termicin polyclonal anti-serum, diluted 1:250 (A) and with preimmune serum at the same concentration (B). As indicated by immunostaining, termicin is localized within blood cell granules. *Bar*, 10 µm.</figcaption></figure>

<table><caption>**FIG. 6.** Comparison of the amino acid sequence of termicin with some members of the insect defensin family. Termicin was compared with defensin A from *Phormia terranovae*, with sapecins A and B from the dipteran *S. peregrina*, and with the Odonate *Aeschna cyanea* defensin. The sequences were aligned for similarities, and gaps are introduced for optimal alignment. Conserved residues between termicin and the insect defensins are in *boldface type*. For *P. terranovae* defensin, the amino acids included in the loop, α-helix, and β-sheets are indicated by *bars*.</caption><tr><td><b><i>Pseudacanthotermes spiniger</i></b></td><td>Termicin</td><td>-A-CNF-Q------S-CWATCQAQHSIYFRRAFCD---RSQCKCVFVRG</td></tr><tr><td><b><i>Phormia terranovae</i></b></td><td>Defensin A</td><td>-ATCDLLSGTGINHSACAAHCL---LRGNRGGYCNG--KGVCVC---RN</td></tr><tr><td><b><i>Sarcophaga peregrina</i></b></td><td>Sapecin A</td><td>-ATCDLLSGTGINHSACAAHCL---LRGNRGGYCNG--KAVCVC---RN</td></tr><tr><td><b><i>Sarcophaga peregrina</i></b></td><td>Sapecin B</td><td>-LTCE-ID-----RSLCLLHCR---LKGYLRAYCSQ-QK-VCRCVQ</td></tr><tr><td><b><i>Aeschna cyanea</i></b></td><td>Defensin</td><td>GFGCPLDQ------MQCHRHCQ--TITGRSGGYCSGPLKLTCTCY--R</td></tr></table>

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 excluded 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 α-helical structure like that of insect cecropins. This prompts a comparison between the biological properties of spinigerin and two representatives of the amphipathic α-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 α-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.
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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