Pherokine-2 and -3 Two Drosophila molecules related to pheromone/odor-binding proteins induced by viral and bacterial infections Laurence Sabatier 1, *, Emmanuelle Jouanguy 1, *†, Catherine Dostert 1 , Daniel Zachary 1 , Jean-Luc Dimarcq 2 , Philippe Bulet 1, ‡ and Jean-Luc Imler 1 1 CNRS UPR9022, Institut de Biologie Mole ´ culaire et Cellulaire, Strasbourg, France; 2 Entomed SA; Illkirch, France Drosophila is a powerful model system to study the regula- tory and effector mechanisms of innate immunity. To iden- tify molecules induced in the course of viral infection in this insect, we have developed a model based on intrathoracic injection of the picorna-like Drosophila Cvirus(DCV).We have used MALDI-TOF mass spectrometry to compare the hemolymph of DCV infected flies and control flies. By contrast with the strong humoral response triggered by injection of bacteria or fungal spores, we have identified only one molecule induced in the hemolymph of virus infected flies. This molecule, pherokine-2 (Phk-2), is related to OS-D/ A10 (Phk-1), which was previously characterized as a putative odor/pheromone binding protein specifically expressed in antennae. The virus-induced molecule is also similar to the product of the gene CG9358 (Phk-3), which is induced by septic injury. Both Phk-2 and Phk-3 are strongly expressed during metamorphosis, suggesting that they may participate in tissue-remodeling. Keywords: host-defense; antiviral; Drosophila C virus; odor- binding protein; tissue remodelling. Innate immunity enables multicellular organisms to detect and fight infectious microbes. In vertebrates, the innate immune system also participates in the induction and shaping of the subsequent adaptive immune response carried by lymphocytes. The innate immune system involves pattern recognition receptors (PRRs) which recognize conserved molecular patterns from broad classes of microorganisms, such as lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan (PGN) from Gram- positive bacteria, or double-stranded (ds)RNA from viruses. Activation of PRRs triggers a host response to control the infection either by acting directly on the microorganisms by phagocytosis or the production of toxic compounds such as nitric oxide and antimicrobial peptides, or by inducing the production of cytokines or costimulatory molecules (reviewed in [1–3]). The fruitfly Drosophila melanogaster is a good model to decipher the molecular mechanisms governing innate immunity in animals because of its well-characterized genetics, and its lack of an adaptive immune system. The significant progress in our understanding of the response of Drosophila to infection by bacteria and fungi made in the past years have revealed a number of molecular similarities between the pathways regulating innate host-defense in flies and mammals [4,5]. The best characterized aspect of the Drosophila response to infection is the inducible synthesis and secretion in the hemolymph of a cocktail of potent antimicrobial peptides active against bacteria and/or fungi. Transcriptional induction of the genes encoding these peptides involves two pathways: infections by fungi and Gram-positive bacteria trigger the Toll pathway, named after the transmembrane receptor Toll, whereas infections by Gram-negative bacteria activate the Imd pathway, named after the immune deficiency (imd) gene. The Toll and Imd pathways exhibit similarities with the interleukin-1 and the TNFa pathways, respectively [4,5]. Following the demonstration of the critical role played by the Toll receptor in Drosophila, a family of related molecules was identified in mammals. These Toll-like receptors (TLRs) are involved in cell activation by microbial molecules such as LPS (TLR4), PGN (TLR2) or bacterial DNA (TLR9) [6,7]. By contrast, nothing is known about the response to virus infection in Drosophila. In mammals, dsRNA from viruses has long been known to activate enzymes such as protein kinase R (PKR) or oligo A 2.5 synthase, and cytokines such as interferon-b. However, our understanding of the mechanisms operating during the innate antiviral response remain sketchy, as illustrated by the recent identification of TLR3 as a transmembrane receptor for dsRNA [8]. In order to analyze the Drosophila host-defense against viral infection, we developed a model based on Correspondence to J L. Imler, CNRS UPR9022, Institut de Biologie Mole ´ culaire et Cellulaire, 15 rue Rene ´ Descartes, 67000 Strasbourg, France. Fax: + 33 388 60 69 22, Tel.: + 33 388 41 70 36 E-mail: JL.Imler@ibmc.u-strasbg.fr Abbreviations:DCV,Drosophila Cvirus;PRR,patternrecognition receptors; LPS, lipopolysaccharide; PGN, peptidoglycan; TLR, Toll-like receptors. *These two authors contributed equally to the work. Present address: INSERM U550; 156 rue de Vaugirard; 75015 Paris, France. àPresent address: Atheris laboratories; Case Postale 314; CH-1233 Bernex, Switzerland. (Received 28 April 2003, revised 12 June 2003, accepted 18 June 2003) Eur. J. Biochem. 270, 3398–3407 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03725.x Drosophila C virus (DCV). DCV is a nonenveloped small single stranded (+) RNA virus, which differs from picornaviridae by its specific genome organization, and the presence of two open reading frames [9]. Drosophila is a natural host for DCV, which is transmitted horizontally through contact or ingestion [10–12]. In a first step to characterize the response of Drosophila to virus infection, we attempted to identify molecules induced by DCV infection that could serve as markers of the antiviral response. We used MALDI-TOF mass spectrometry (MS) differential analysis between the hemolymph of DCV- infected flies vs. unchallenged Drosophila. This differential MS approach was developed by Uttenweiler-Joseph and colleagues to study the antibacterial response, and allowed the identification of more than 24 small peptides, named Drosophila immune-induced molecules [13,14]. Using the same approach, we have identified only one peptide which is induced upon virus infection. This peptide presents strong sequence similarity to OS-D/A10, a molecule previously characterized as a putative odor-binding protein [15,16]. A third Drosophila molecule belonging to this small family is induced by septic injury. Experimental procedures Plasmids The attacin A promoter in the pCasper transformation vector pJL166 [17] was replaced by a XbaI–NheIPCR fragment containing 2.6 kb of phk-2 5¢ untranslated sequences (GenBank AE003462 nt250436–253041) to obtain pJL265. This fragment includes exon 0 of phk-2, the first intron, and the first exon to the ATG, which is used to initiate translation of GFP S65T. The transforming vector pJL264 contains a shorter fragment of phk-2 5¢ untranslated sequences (GenBank AE003462 nt2516916– 253041), and yielded identical results (data not shown). The phk-2 cDNA (EST clone GH24283) was obtained from the Berkeley Drosophila Genome Project (Berkeley, CA; http:// www.fruitfly.org). The phk-2 cDNA was subcloned as an EcoRI–XhoI fragment between the corresponding sites in the pP{UAST} vector [18]. Fly strains and bacterial infections Oregon-R and ywDD1; cnbw wild-type flies were used throughout this study [19]. Flies were maintained on a standard cornmeal medium at 25 °C. Transgenic lines were generated by P element transformation of a w – strain. Standard crosses with flies carrying appropriate balancers were performed to establish stable heterozygous or homo- zygous lines, as well as to determine the chromosome carrying the insertion. At least three independent lines were analyzed for each construct. To overexpress Phk-2, females carrying the UAS-phk-2 transgene were crossed with males carrying the P{GAL4-YP1.JMR} (yolk protein 1 gene promoter-Gal4) driver [20]. Bacterial infections were per- formed by pricking adult flies with a thin needle, previously dipped in a concentrated culture of Escherichia coli and Micrococcus luteus. RNA extraction, Northern blot analysis and RT-PCR on total RNA were performed as described previously [21]. Preparation of the DCV stock An isolate of DCV was kindly provided by X. Jousset and M. Bergoin (INRA-CNRS URA2209, St Christol-Lez- Ale ` s, France). A concentrated viral suspension was prepared by successive rounds of amplification in infected adult flies. This was purified on a caesium chloride gradient and analyzed by electron microscopy as described [22]. Briefly, 4000 flies were injected with DCV and collected and frozen after death. Flies were crushed in 10 m M Tris/HCl (pH 7.5), followed by sonication (20 kHz; three times for 5 s). The extract was deposited on a 20% (w/v) sucrose solution, and centrifuged (25 000 g,1h30min,15°C).Theviralpellet was resuspended in 10 m M Tris/HCl (pH 7.5). After sonication as above, the viral suspension was added to a tube containing two layers of caesium chloride (5% and 40%), and ultracentrifuged for 16 h at 36 000 g at 15 °C. The virus band was collected and DCV was recovered by centrifugation (25 000 g,1h30min,15°C). The purified viral pellet was resuspended in 1 mL 10 m M Tris/HCl (pH 7.5), sonicated as above, aliquoted and stored at )80 °C. The viral titer was estimated to be 10 11.5 LD 50 ÆmL )1 , using the Reed–Muench endpoint method and a 7 day incubation period in adult flies [23]. For the infection experiments described, 5 nL containing 10 4.5 LD 50 were injected in the thorax of 4–6-day-old adult flies. For survival experiments, groups of 25 flies were kept on standard medium at 22 °C, and counted daily. Microscopic observations For observation of GFP expression patterns, live flies and larvae were anaesthetized with ether or on ice, and viewed under epifluorescent illumination (excitation filter, 480 nm; dichroic filter, 505 nm; and emission filter, 510 nm) with a Leica MZFLIII dissecting microscope and images were recorded using a digital charge-coupled device Spot RT color camera (Diagnostic Instruments). For histology analysis, dissected female flies were fixed in 4% (v/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 1 h at 4 °C, postfixed with osmium tetroxide, embedded in araldite/epon, and sectioned for optic or electron microscopy. A toluidin blue coloration was performed on semithin sections. Briefly, after a 2 min treatment with sodium methoxide, the sections were incubated in a 50% methanol/benzene mixture (v/v) during 90 s, followed by acetone for 1 min, before rinsing in distilled water. The slides were then stained for 5 min in a toluidin blue solution (0.1% toluidin blue, 1% borax; pH 11), rinsed with distilled water and dehydrated. For transmission electron micro- scopy, preparations of dissected tissues were fixed in 4% (v/ v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 1 h at 4 °C, and postfixed with osmium tetroxide. Samples embedded in araldite/epon were sectioned and counterstained with lead citrate and uranyl acetate. MALDI-TOF MS analysis For mass spectrometry analysis, hemolymph of DCV-, or buffer-injected Drosophila was collected and directly deposited on the target. Samples were prepared according to the sandwich method using the matrix Ó FEBS 2003 Drosophila humoral response to virus infection (Eur. J. Biochem. 270) 3399 a-cyano-4-hydroxycinnamic acid [24]. MALDI-TOF mass spectrometry was performed with a Bruker BIFLEX III TM (Bremen, Germany) mass spectrometer operating in a positive linear mode using an external calibration and synthetic peptides (MH + 2199.6; 3046.4; 4890.5). Purification of Phk-2 Hemolymph from 430 flies was collected in 0.1% (v/v) trifluoroacetic acid 72 h after DCV infection. After centri- fugation (10 000 g, 20 min), the supernatant was subjected to gel permeation HPLC using two serially linked columns (Ultraspherogel SEC 3000 and SEC 2000 columns, 7.5 · 300 mm, Beckman). Elution was performed under isocratic conditions with 30% acetonitrile in 0.05% (v/v) trifluoroacetic acid at a flow rate of 0.4 mLÆmin )1 . Fractions were hand-collected according to the absorbance at 225 nm and analyzed by MALDI-TOF mass spectrometry. The fraction containing the induced molecule Phk-2 was further purified by reverse phase HPLC on a microbore Aquapore RP300 C 8 column (1 · 100 mm, Brownlee TM Perkin Elmer) using a linear biphasic gradient of acetonitrile in 0.05% (v/v) trifluoroacetic acid from 2 to 25% over 10 min and from 25 to 35% over 50 min, at a flow rate of 80 lLÆmin )1 . Structure identification Purified Phk-2 was treated with trypsin (modified sequen- cing grade, Roche Diagnostics, Mannheim, Germany) using the conditions recommended by the supplier. Digestion was carried out at 37 °Cfor16hin0.1 M Tris/HCl pH 8.9 supplemented with 10% (v/v) aceto- nitrile. The reaction was stopped by acidification and the peptide fragments were separated on a capillary FUS- 15–03-C18-PepMap column (0.3 · 150 mm, LC Packings, Amsterdam, the Netherlands) using a linear gradient of acetonitrile in 0.05% (v/v) trifluoroacetic acid from 5 to 40% over 40 min at a flow rate of 4 lLÆmin )1 at the temperature of 30 °C. The column effluent was monitored by absorbance at 214 nm and the fractions were hand- collected and analyzed by MALDI-TOF MS. Three purified fragments were submitted to automated Edman degradation on a pulse liquid automatic sequenator (Applied Biosystems Model Procise cLC). Cell culture experiments S2 cells were purchased from Invitrogen, and maintained in Schneider’s medium supplemented with 10% (v/v) fetal bovine serum; 60 mgÆL )1 penicillin and 100 mgÆL )1 strepto- mycin. 20-Hydroxyecdysone was added to the cells (10 )6 M ) 48 h prior to stimulation with 10 lgÆmL )1 LPS (E. coli serotype 055:B5, Sigma). Results Systemic infection of Drosophila by DCV We prepared a stock of DCV by serial passages in flies (see Experimental procedures). When aliquots of this suspension were injected into flies, we observed a rapid lethality that was dose-dependent (Fig. 1A). Histological analysis revealed morphological defects associated with the fat body appearing two to three days postinfection as cells started to shrink and stained more strongly with toluidin blue (Fig. 1B,D). Similar changes were detected in cells of the perioveolar epithelial sheath. Consistent with these obser- vations, transmission electron microscopy revealed high levels of DCV particles in cells from the fat body and the perioveolar sheath (Fig. 1E,F). Virus particles were also detected in cells from tracheae, muscles and the digestive tract. The quantity of virus present in these tissues increased over time, indicating that productive infection had occurred (data not shown). DCV infection triggers a discrete humoral response We have analyzed the hemolymph of single flies injected with a suspension of DCV or Tris buffer through a differential display analysis by MALDI-TOF MS. We did not observe induction of any molecules in the 0–10 kDa size range in the mass fingerprint, at any of the time points analyzed (24 h, 48 h, 72 h; Fig. 2A and data not shown). In particular, the antimicrobial peptides which rapidly appear in the hemolymph upon bacterial or fungal infections [14] are not present in the hemolymph of DCV-infected flies. However, one molecule at a measured average molecular mass of 12 820 Da is clearly induced in the hemolymph of flies infected with the virus 48 h after the beginning of the infection (Fig. 2A). This molecule is not present in the hemolymph of flies injected with buffer, or challenged by septic injury with a mixture of Gram-negative and Gram- positive bacteria, or upon natural infection with the entomopathogenic fungus Beauveria bassiana (data not shown and [14]). Hemolymph from DCV-injected flies was analyzed by HPLC and the molecule induced by the viral infection was detected by MALDI-TOF MS (Fig. 2B). The molecule was purified to homogeneity by gel permeation and reversed-phase chromatography and submitted to proteolysis for structural characterization. The digest was purified, and the recovered fragments sequenced by Edman degradation. Database analysis using one of these sequen- ces, namely YIIENKPEEWK, revealed that the virus- induced molecule corresponds to the product of the gene PebIII [25] (also called CG11390; http://www.fruitfly.org/) (Fig. 2C). The Drosophila genome contains two additional genes related to PebIII/CG11390 (Fig. 2D): OS-D/A10 has been described previously on the basis of its tissue-specific expression in the olfactory region of antennae [15,16]; CG9358 has not been described previously. Interestingly, expression of the latter gene appears to be upregulated in response to bacterial or fungal infections [26–28] (see below). We therefore propose to call these molecules induced by infection which may function as odor/phero- mone binding proteins pherokines (Phk-1, OS-D/A10; Phk-2, PebIII/CG11390; Phk-3, CG9358). Sequencing of the fragment with a measured mass of MH + 1170.9 showed a difference (Asp/Glu) between the sequence from the Drosophila genome (CG11390) and the sequence of Phk-2 (Fig. 2C). The start of the mature protein after cleavage of the signal peptide was ascertained by the sequencing of the N-terminal peptide (measured mass MH + 2204.2). The concentration of Phk-2 in the hemo- lymph of DCV-infected Drosophila was estimated at 40 n M . 3400 L. Sabatier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 1. Systemic infection following intrathoracic injection of DCV in Drosophila. (A) Adult flies (Oregon-R) were injected with 5 nL of different dilutions of a DCV stock (10 11.5 LD 50 ÆmL )1 ), or with buffer (Tris). Surviving flies were counted daily. (B–D) Histology of the fat body of flies 2 (C) or 4 (B,D) days after injection of Tris (B) or DCV (C,D). The scale bar represents 20 lm (E,F). Accumulation of viral particles in the cytoplasm of cells from the fat body (E) or the perioveolar sheath (F) 4 days after injection of DCV. Crystal-like arrangements of virus particles are indicated by stars and enlarged in insets. The scale bars represent 0.3 lminEand0.5lm in F; m, mitochondria; n, nucleus; ov, ovary; ch, chorion. Ó FEBS 2003 Drosophila humoral response to virus infection (Eur. J. Biochem. 270) 3401 Phk-2 is regulated by DCV infection at the post-transcriptional level Northern blot analysis revealed that the quantity of transcripts encoding Phk-2 does not increase following viral infection (data not shown). This result suggests either that phk-2 is not regulated at the transcriptional level, or that the induction of phk-2 in some tissues is masked by constitutive expression in others. To identify the expression domains of this gene, we constructed transgenic strains of Drosophila expressing GFP under the control of 2.2 kb of 5¢ untranscribed sequences from phk-2. We observed green fluorescence in noninfected larvae in both anterior and posterior spiracles, in the digestive tract, the ring gland, the antenna buds and testis (Fig. 3A,B and data not shown). In adult flies, GFP was expressed in several tissues, including the legs, the wing veins, the male and female reproductive tracts, the digestive tract and the labellum (Fig. 3C–F). Analysis by mass spectrometry, RT-PCR (Fig. 3G–I), and in situ hybridization [25] confirmed that the endogenous gene is expressed in fluorescent tissues. These results are sum- marized in Fig. 3J,K. Importantly, we did not observe any modification of the fluorescence pattern in DCV infected flies, confirming that Phk-2 induction by DCV infection is not mediated at the transcriptional level (data not shown). Constitutive production of Phk-2 does not protect flies from DCV infection In the absence of mutant strains for the phk-2 gene, we constructed strains of Drosophila constitutively expressing Phk-2 in the hemolymph, using the UAS-Gal4 system [18]. Fig. 2. Identification of Phk-2 in the hemo- lymph of DCV infected flies. (A) MALDI-TOF mass spectrometry analysis of the hemolymph of single flies 3 days after injection of buffer (Tris) or a DCV suspension. The position of the 12 820 Da DCV-induced molecule (Phk- 2) is indicated. The peaks present in the Tris- injected fly but not in the DCV-injected fly are not reproducibly observed and correspond to previously described Drosophila induced mole- cules transiently upregulated following injury [14]. (B) HPLC chromatography of the hemolymph of 40 flies 3 days after injection of a DCV suspension. The column is a microbore Aquapore RP300 C 8 column (1 · 100 mm, Brownlee Labs) eluted with a linear gradient of acetonitrile (2–80%, v/v) in acidified water at a flow rate of 80 lLÆmin )1 at 35 °C. The position of Phk-2, detected by MALDI-TOF mass spectrometry, is indicated. (C) Sequence of the cDNA clone GH24283 which encodes Phk-2. The amino acid sequence is shown below the nucleotide sequence. The sequences of the tryptic peptides sequenced after purifi- cation of Phk-2 are underlined. The only difference between these sequences is the replacementofanasparticacidatposition108 by a glutamic acid. The signal peptide sequence is boxed, and the two disulfide bridges are indicated as determined experi- mentally. (D) Alignment of the mature sequences of Drosophila pherokines with related molecules from other insects. 3402 L. Sabatier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Overexpression of phk-2 using the ubiquitous drivers daughterless-Gal4 and actin5C-Gal4 or the fat body specific driver yolk-Gal4, was not lethal and did not induce any obvious phenotype (data not shown). Phk-2 was constitu- tively present in the hemolymph of female flies expressing both the yolk-Gal4 driver and the UAS-phk-2 transgene (Fig. 4A). However, susceptibility of these flies to DCV infection did not significantly differ from that of control flies (Fig. 4B). Regulation of pherokine expression during infection and development We confirmed by Northern blot analysis that the quantity of phk-3 transcripts increases in response to septic injury, with a peak at 3–6 h postinfection (Fig. 5A). Under the same conditions, phk-1 and phk-2 are not upregulated. phk-3 remains inducible in Dif, dorsal, spaetzle and kenny mutant flies [4], suggesting that it is not regulated by the Toll or Imd Fig. 3. Expression pattern of phk-2. (A,B) phk-2-GFP transgenic larvae exhibit green fluorescence in the ganglia of the antenno-maxillary organ (A, arrow), in the anterior (A) and posterior (B) spiracles (arrowheads), in the ring gland (A, asterisk) and in the hindgut (B, dot). (C–F) GFP expression in the legs (C), wing veins (D) and reproductive tract (E,F) of phk-2-GFP transgenic flies. In males (E), GFP is expressed in the ejaculatory bulb (arrow) and restricted areas of the seminal vesicles (arrowheads). In females (F), green fluorescence can be detected in the uterus (arrow) and seminal receptacle (arrowhead). The hindgut and rectum are shown in bracket (E,F). (G,H) MALDI-TOF mass spectrometry analysis of dissected ejaculatory bulb (G) and legs (H) showing expression of a 12.8 kDa molecule (arrow). (I) Fluorescent (hindgut) and nonfluorescent (midgut) parts of the gut of phk-2-GFP transgenic flies were dissected, and used to extract mRNA. Expression of rp49 and phk-2 was monitored by RT-PCR. For rp49, 25 cycles of amplification were performed. For phk-2, 35 cycles of PCR were performed, followed by 25 cycles on an aliquot (2 lL) of this reaction with nested primers. (J,K) Summary of the expression pattern of phk-2 in noninfected larvae and adult flies. Ó FEBS 2003 Drosophila humoral response to virus infection (Eur. J. Biochem. 270) 3403 pathways (Fig. 5B and data not shown). phk-3 is the only member of the family expressed in the macrophage-like S2 cells. Furthermore, its expression is upregulated upon treatment with LPS (Fig. 5C). Pherokines also display interesting developmental expression patterns. Expression of phk-3 is first detectable at the end of embryogenesis and in larvae, and the highest expression level is observed in white pupae (0–72 h). Expression decreases in black pupae (72–96 h) and adults. phk-2 is also expressed in larvae and pupae, with a strong peak of expression in black pupae. Both phk-2 and phk-3 are more strongly expressed in male than female adult flies. Finally, phk-1 expression starts in black pupae, when the development of olfactory sensilla is essentially complete, and remains constant in adult flies (Fig. 5D). Consistent with this regulated expression pattern during metamorphosis, we observed that treatment of S2 cells with the molting steroid hormone 20-hydroxyecdysone completely suppresses phk-3 expression (Fig. 5C). By con- trast, 20-hydroxyecdysone treatment strongly potentiates the immune-inducibility of the gene encoding the antibac- terial peptide Diptericin, as described previously [21]. Discussion Antiviral response in Drosophila Our results reveal striking differences in the response of Drosophila to infection with the virus DCV compared to bacteria or fungi. Indeed, one hallmark of the response to bacterial or fungal infections is the inducible secretion into the hemolymph of a cocktail of antimicrobial peptides [4,5]. In addition, a large number of Drosophila immune-induced molecules are also induced in the hemolymph following septic injury [13,14,29]. By contrast, none of these molecules are induced upon DCV infection at the time points analyzed, and we only identified a single induced molecule, Phk-2, in the hemolymph of DCV-infected flies. Constitu- tive overexpression of Phk-2 does not protect flies against a DCV challenge, suggesting that it is not directly involved in the antiviral response. Rather, this molecule may be involved in tissue-repair, or in the behavior of infected flies (see below). In agreement with these biochemical data, we observed that Dif (Toll pathway) and key (Imd pathway) mutant flies exhibit the same sensitivity to DCV infection as wild-type flies (data not shown). Importantly, bacterial challenge 48 h after the injection of DCV led to normal induction of antimicrobial peptide genes, indicating that the Toll and Imd pathways in fat body cells are not affected by DCV infection, at least in the first three to four days of infection. Altogether, these experiments suggest that the host-defense mechanisms against virus infection are differ- ent from the mechanisms operating during bacterial or fungal infections in flies. In future work, it will be interesting to compare the response of Drosophila to other types of viruses such as Sigma virus [30], to confirm that the pathways regulating antibacterial and antifungal responses Fig. 4. Constitutive expression of Phk-2 in the hemolymph of transgenic flies is not sufficient to protect them against DCV infection. (A) MALDI- TOF mass spectrometry analysis of the hemolymph from a single female fly containing the UAS-phk-2 transgene and the fat-body spe- cific yolk-Gal4 driver (left panel). The analysis of the hemolymph from a control female fly containing the UAS-phk-2 transgene but not the driver is shown in the right panel. (B) Flies of the indicated genotypes and gender (the yolk promoter is only active in the fat body of female flies) were infected with DCV (10 4.5 LD 50 ), and survival was moni- tored daily. Two independent experiments are shown. Flies constitu- tively expressing Phk-2 in the hemolymph are indicated with squares, andcontrolflieswithcircles.Transgeniclineswereestablishedinaw – (w) background (see Experimental procedures). Note that the genetic background of these flies differs from the Oregon-R flies used in Fig. 1, which explains the different susceptibility to DCV infection (our unpublished data). Fig. 5. Expression of phk genes in response to infection and during development. (A) phk-3 Transcripts are transiently upregulated fol- lowing infection with a mixture of Gram-positive and Gram-negative bacteria. Drosomycin was used as a positive control and rp49 as a loading control. (B) ywDD1; cnbw wild-type flies (WT), Dif (Toll pathway) and key (Imd pathway) mutant flies were infected by septic injury, and expression of phk genes was analyzed by Northern blot. (C) phk-3 Expression is upregulated by LPS (+) and repressed by the molting hormone ecdysone in S2 tissue-culture cells. (D) Develop- mental expression profile of pherokines. Poly(A)+ RNA was extrac- ted from embryos, third instar larvae, L(3), 0–72 h white pupae (w), 72–96 h black pupae (b), and male or female adults, and analyzed by Northern blot using the indicated probes. 3404 L. Sabatier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 differ from those activated by viral infection. It will also be interesting to study flies infected through the respiratory or digestive tracts [12]. Pherokines and chemosensation We describe in this report two new molecules which are induced by septic injury. Phk-2 is induced by DCV infection, whereas Phk-3 is induced by bacterial challenge. The third member of this family in Drosophila, Phk-1, is not induced and is specifically expressed in the olfactory segment of antennae [15,16]. Pherokines belong to a family of small hydrophilic secreted peptides isolated from several insect species on the basis of their tissue-specific expression in the olfactory organs (e.g. [31–34]). They are characterized by four cysteines involved in two disulfide bridges and forming a CX 6 CX 18 CX 2 C signature motif, and differ from the mem- bers of the major family of odorant-binding proteins in Drosophila, which are characterized by six conserved cysteine residues [35]. Based on their tissue-specific expression, these molecules have been suggested to participate in sensing odors and/or pheromones. Our data thus raise the provocative prospect that the sensorial system may play a role in host- defense in Drosophila, as previously reported in social insects [36,37]. In another invertebrate, Caenorhabditis elegans,the Toll receptor CeTol-1 was recently shown to participate in chemosensory behavior, enabling worms to avoid ingestion of pathogenic bacteria [38]. Pherokines may be involved in a similar type of chemosensory behavior in Drosophila. Another interesting possibility is that pherokines may participate in the control of reproduction in Drosophila. Indeed, the cabbage armyworm Mamestra brassicae mole- cule MbraAOBP2, which shares 50% identity with Phk-2, has been shown to bind the pheromone vaccenyl acetate [32]. Interestingly, phk-2 is expressed in the ejaculatory bulb of Drosophila males, which contains cis-vaccenyl-acetate. This pheromone is transferred by males to females during copulation, and has an antiaphrodisiac effect on male courtship [39]. Phk-2 may act as a carrier in this process. Thus, the induction of Phk-2 by DCV infection may be connected to modification of the fly’s reproduction dynam- ics. This could represent an efficient host-defense strategy, as DCV is not transmitted vertically. Importantly, DCV- infected flies have been shown to have higher fecundity and fertility than DCV-free animals [10,12]. However, we have so far failed to detect changes in the reproductive dynamics of flies overexpressing phk-2 (data not shown). Pherokines and host-defense The fact that one member of the family, namely Phk-1, may function as an odor/pheromone-binding factor does not necessarily imply that the other members exhibit similar functions. In agreement with this possibility, we found that phk-2 is expressed in many tissues not linked to chemo- sensory functions. There is at least one report in which a pherokine-related molecule has been isolated in a context different from olfaction. In the larval stage, the cockroach Periplaneta americana can regenerate lost tissues or organs such as the eyes, the antennae or the legs. The P. americana protein p10, which shares 50% identity with Phk-2, is strongly and transiently upregulated during the regeneration of the legs in larvae [40]. Thus pherokines may have a general role in tissue remodeling in response to injury or in a developmental context. In keeping with this hypothesis, we have shown that the phk-2 and phk-3 genes are highly expressed during metamorphosis in Drosophila. In addition, we have shown that the phk-2 promoter is active in the ring gland in larvae, a neuroendocrine center which produces the hormones controlling molting, metamorphosis, reproduc- tion and organ growth. Finally, our finding that phk-3 is downregulated by ecdysone treatment in S2 cells was recently confirmed in a genome-wide analysis of steroid-induced cell death which showed that expression of both phk-2 and phk-3 is strongly reduced by ecdysone in vivo [41]. These data support the hypothesis that Phk-2 and Phk-3 may interact with ligands different from Phk-1, and carry other functions than chemosensation. Similar observations were made in mammals, where some odor-binding proteins, which are specifically expressed in olfactory epithelia, are structurally related to molecules involved in the binding and transport of other molecules. This is the case, for example, for OBP, which belongs to the same structural family as the retinol-binding protein and the cholesterol-binding protein apoD [42], or of RYA3, which exhibits significant sequence homology to the LPS-binding protein [43]. This latter example finally raises the possibility that all pherokines, including Phk-1, serve a primary defense function by recognizing and/or neutralizing invading microorganisms. The openings of the chemosen- sory sensillae clearly represent an easy entry for microbes, and mechanisms to maintain sterility of the sensillar fluid are likely to exist, possibly including expression of phk-1.The fact that the antenno–maxillary complex, which mediates olfaction in larvae, expresses two antimicrobial peptides upon exposure of larvae to bacteria confirms the existence of host-defense mechanisms associated with olfactory tissues in Drosophila [17]. In summary, we have identified a family of molecules that are expressed in a regulated manner during infection and development. Some members of this family are expressed in a tissue-specific manner in olfactory organs, where they may function as odor- or pheromone-binding molecules. Other members may function as ligand-binding molecules for other factors regulating tissue repair or remodeling. Future studies using the powerful genetics of Drosophila will help to clarify the exact physiological roles of pherokines. Our data further suggest that the response to virus infection involves mechanisms different from those operating to control bacterial or fungal infections. Acknowledgements WewouldliketothankRene ´ Lanot for help with the microscopy analysis; Estelle Santiago for expert technical assistance; Sebahat Ozkan for help with transgenesis; Xavie ` re Jousset and Max Bergoin for providing virus stocks and much useful advice in the early stages of this project and Liliane Gloeckler and Anne-Marie Aubertin for assistance in producing virus stocks; Dominique Ferrandon and Jules Hoffmann for critical reading of the manuscript and stimulating discussions. This project was funded by CNRS, Entomed, as well as a grant from the Ministe ` re de la Recherche et de la Technologie (ACI Physiologie Inte ´ grative). EJ was supported by a postdoctoral fellowship from the Ligue contre le Cancer. CD is supported by a fellowship from the Ministe ` re de la Recherche du Grand-Duche ´ de Luxembourg. Ó FEBS 2003 Drosophila humoral response to virus infection (Eur. J. Biochem. 270) 3405 References 1. Girardin, S.E., Sansonetti, P.J. & Philpott, D.J. (2002) Intracellular vs extracellular recognition of pathogens – common concepts in mammals and flies. Trends Microbiol. 10, 193–199. 2. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A. & Ezekowitz, R.A. (1999) Phylogenetic perspectives in innate immunity. Science 284, 1313–1318. 3. Kimbrell, D.A. & Beutler, B. (2001) The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267. 4. Hoffmann, J.A. & Reichhart, J.M. (2002) Drosophila innate immunity: an evolutionary perspective. Nat. 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