Peptidomic and proteomic analyses of the systemic immune response of Drosophila

Biochimie 86 (2004) 607–616 www.elsevier.com/locate/biochi

Peptidomic and proteomic analyses of the systemic immune response of Drosophila Francine Levy, David Rabel, Maurice Charlet, Philippe Bulet 1, Jules A. Hoffmann, Laurence Ehret-Sabatier * Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg cedex, France Received 19 April 2004; accepted 6 July 2004 Available online 09 August 2004

Abstract Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine a-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila. © 2004 Elsevier SAS. All rights reserved. Keywords: Drosophila hemolymph; Innate immunity; Differential analysis; Mass spectrometry; 2D electrophoresis

1. Introduction Innate immunity is the first line of defense against invading pathogens. From invertebrates to vertebrates, characteAbbreviations: 2D, two-dimensional; AMP, antimicrobial peptide; CBB, Coomassie brilliant blue; DIM, Drosophila immune-induced molecule; GNBP, Gram-negative binding protein; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; MALDI-TOF, matrixassisted laser desorption/ionization time-of-flight; MS, mass spectrometry; MPAC, maturated pro-domain of attacin C; PGRP, peptidoglycan recognition protein; TEP, thioester-containing protein; Tsf, transferrin; OBP, odorant binding protein; PEBP, phosphatidylethanolamine binding protein. * Corresponding author. Tel.: +33-388-41-70-62; fax: +33-388-60-69-22. E-mail address: [email protected] (L. Ehret-Sabatier). 1 Present address. Atheris Laboratories, Case Postale 314, CH-1233 Bernex, Geneva, Switzerland. 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.07.007

rization of innate immune mechanisms in multicellular organisms revealed striking similarities suggesting a common evolutionary ancestry [1]. With its particularly convenient genetics and fully sequenced genome, the fruit fly, Drosophila melanogaster, has emerged as a powerful model system to study these first line defense mechanisms. To combat microbial infections, Drosophila has developed a large range of strategies based on cellular and humoral reactions. This includes phagocytosis by macrophage-like blood cells, activation of proteolytic cascades leading to localized melanization and coagulation, and synthesis of potent antimicrobial peptides (AMPs) by the fat body. Drosophila AMPs are grouped into three families based either on their main antimicrobial activity in vitro or on the activities reported for related peptides isolated from other insects. The first family corresponds to peptides active preferentially on Gram-

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negative bacteria: drosocin, cecropin, diptericin and attacin. The second family corresponds to the defensin, active mainly against Gram-positive bacteria. Finally, the last group of peptides consists of drosomycin and metchnikowin that are active exclusively against fungi. Two distinct signaling pathways, Toll and immune deficiency (Imd), have been shown to control the expression of these AMPs. The Toll pathway participates in the defense against infections by fungi and Gram-positive bacteria, while an infection with Gramnegative bacteria activates the Imd pathway (reviewed in [2,3]). Molecular and genetic studies have allowed the partial characterization of these two pathways, both of which involve members of the Rel family of transcription factors. Recently, it was shown that peptidoglycan recognition proteins (PGRPs) are involved in the recognition of microbes in the Toll and Imd pathways [4,5]. A key point of the Toll pathway is an extracellular proteolytic cascade leading to the cleavage of the protein Spaetzle, the ligand of the transmembrane receptor Toll [6]. In the case of fungal activation, the cascade is negatively regulated by the blood serpin Necrotic (Nec, [7]) which inhibits the serine protease Persephone (Psh, [8]). On the other hand, Toll activation by Grampositive bacteria involves two extracellular proteins thought to form a complex after the infection, PGRP-SA [4] and a Gram-negative binding protein, GNBP 1 [9]. Although much has been learnt about Drosophila immunity through genetic studies many aspects remain unclear, in particular the identity of the molecules that are recruited or activated in the hemolymph and involved in the signalization cascades. With the increase of genome sequencing, transcriptomics has proved to be a powerful approach for functional analysis of gene products. Nevertheless, several groups have reported a poor correlation between mRNA levels and protein abundance in the cell [10,11], indicating that there is a clear interest in directly monitoring peptide and protein levels. It is also important to remember that most proteins undergo posttranslational modifications that can affect their function. Peptidomic and proteomic strategies have thus emerged as valuable techniques to progress in the post-genomic analysis of Drosophila defense reactions. Differential analyses of hemolymph from immune-induced versus unchallenged Drosophila were performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for molecules with a molecular mass below 15 kDa and two-dimensional (2D) gel electrophoresis for molecules with greater molecular masses. MALDI-TOF differential display led to the detection of 24 Drosophila immune-induced molecules (DIMs) after bacterial or fungal insult [12] and one molecule induced after a viral infection [13]. Combining high performance liquid chromatography (HPLC) and MS led to the detection of additional induced peptides. Whilst some of the DIMs corresponded to molecules already known, drosocin, metchnikowin or drosomycin, the primary structure of others was defined through biochemical approaches. To establish the profile of hemolymph molecules with a molecular mass

Fig. 1. Differential MALDI-TOF MS spectra of hemolymph from a control and an immune-challenged adult Drosophila. Hemolymph was collected from a single adult fly (control or immune-challenged with a mixture of Gram-positive and Gram-negative bacteria) and analyzed by MALDI-TOF MS. The singly charged ions of the molecules induced after immune challenge were numbered from 1 to 24, except for DIM 20, which is the doubly charged ion of DIM 24 mistakenly annotated. Five peaks correspond to known AMPs, which are also annotated by their names.

greater than 15 kDa, a differential proteomic approach was used, combining 2D gel electrophoresis, MS and data bank searches. At least 70 proteins were found to be up- or downregulated after a bacterial or fungal challenge [14]. Among these, proteins from many functional classes such as proteases, serpins or molecules involved in iron metabolism were identified. In this review we describe the molecules present in adult Drosophila hemolymph that were found to be altered after bacterial or fungal challenge through peptidomic and proteomic approaches. 2. Peptidomic approach To investigate molecules induced during a systemic immune reaction, we used a differential approach based on MALDI-TOF MS (Fig. 1). This strategy allowed us to detect 24 DIMs in the hemolymph of immune-challenged versus control flies, with molecular masses ranging from 1666 Da (DIM 1) to 10,031 Da (DIM 24). A time-course analysis showed that all the DIMs are present in the hemolymph from 3 h post-infection. In addition, two phases of induction were observed, one with a maximum concentration at 6 h postinfection (DIMs 1–8, 14, 15, 22 and 23) and the other with a maximum at 24 h post-infection (DIMs 9–13, 16–19 and 24). If we except drosomycin (DIM 19), the concentration of all DIMs strongly decreased 2 days after infection. Analysis of Drosophila mutants showed that the expression of most of these 24 DIMs was regulated by the Toll pathway, except DIMs 9, 11, 15 (drosocins) and DIM 16, which were found to be under control of the Imd pathway. DIM 17 (metchnikowin) can be regulated by both pathways [15]. A combination of HPLC and MALDI-TOF MS led to the detection of additional induced molecules. These and other

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DIMs were purified by size-exclusion and reverse-phase HPLC, then primary structure was established using N- and C-terminal sequencing, proteolytic treatment, tandem mass spectrometry, cDNA cloning and data bank searches. All the DIMs characterized so far are reported in Table 1 and their main characteristics are described below.

O-glycosylated form of diptericin carrying one carbohydrate moiety. It was detected thanks to an anti-Drosophiladiptericin antibody. Following endoproteinase Asn-C cleavage and MALDI-TOF MS analysis, fragments corresponding to native diptericin were detected, as well as potential glycosylated peptides.

2.1. DIMs corresponding or related to known AMPs

2.2. DIMs corresponding to molecules not related to AMPs

DIMs 17, 19, 25 and 26 correspond to AMPs, which have already been described. DIM 17 is metchnikowin, a prolinerich AMP with activity against filamentous fungi; DIM 19 is drosomycin, which has antifungal properties; DIM 25 is cecropin A, which preferentially kills Gram-negative bacteria; DIM 26 corresponds to defensin, which is particularly active against Gram-positive bacteria and moderately effective against some Gram-negative bacterial or filamentous fungal strains (for a review see [16]). DIMs 9 and 11 correspond to drosocin with one and two sugars at Thr11, respectively. They belong to the proline-rich AMP family. These molecules are preferentially active against Gram-negative bacteria [17]. Glycosylated forms of drosocin have been shown to be more active against bacteria than unmodified drosocin [18]. DIM 15 is a third variant of drosocin carrying an additional carbohydrate moiety at Ser7 [19]. DIM 16 was recently identified as a highly posttranslationally-modified, biologically active form of the prodomain of the AMP attacin C (MPAC, [19]). While its sequence has strong similarities with drosocin, MPAC alone has a restricted antimicrobial activity against some Gramnegative bacteria. Interestingly, this activity is potentiated when MPAC is used in synergy with the Drosophila AMP cecropin A, with a minimal active concentration of 3.2 µM for the inhibition of Enterobacter cloacae b12 growth [19]. This was the first documented case where an antibacterial polypeptide was cleaved to form two independent functional antibiotic units. DIMs 21 and 27 are the most recently identified AMPs isoforms. DIM 21 has been proposed to be a posttranslationally modified variant of drosomycin. Edman sequencing of DIM 21 led to the identification of the first 12 residues that are fully identical with drosomycin. Nevertheless, DIM 21 shows a mass difference of approximately 1045 Da more in comparison to that of the originally isolated drosomycin [20,21]. The sequence of drosomycin contains a consensus glycosylation site NET, from residue 16 to residue 18. This site could carry a carbohydrate moiety, which may be responsible for the difference in mass observed between drosomycin and DIM 21 (calculated mass for two N-acetylhexosamine, three hexose and one pentose is 1042 Da). These data led us to propose that DIM 21 corresponds to an N-glycosylated form of drosomycin. Interestingly, the Drosophila genome contains seven drosomycin genes, but only products from one of these genes have been found in adult hemolymph. DIM 27 is thought to be an

DIMs 29 and 30 are cleavage products of larger molecules. DIM 29 corresponds to an N-terminal fragment of a vitellin membrane protein (VM26Ab), while DIM 30 is a fragment of the gene product of vitellogenin III (Yp3). During the Drosophila immune response several proteolytic cascades are activated, involving numerous proteases, which may be responsible for such cleavages. Examples of proteins with structural similarities to vitellogenin have already been implicated in immune mechanisms [22,23]. Interestingly, a fragment of the CG11064 gene product, structurally similar to vitellogenins [24], was recently found to be overexpressed in a 2D differential electrophoretic study of Drosophila larvae hemolymph proteins after lipopolysaccharide (LPS)-stimulation [25]. DIM 33 is a serine protease inhibitor of the Kunitz family. Transcriptional profiling studies found that various genes of this family were over-expressed during a microbial infection and they are suspected to play an important role in innate immune reactions [26,27]. In particular CG16713, whose product displays high similarity with DIM 33, is upregulated after an immune-challenge [27]. It should be emphasized that activation of the Toll pathway is known to be under control of a proteolytic cascade but few of the proteases or protease inhibitors involved have as yet been characterized. The identification of DIM 33 as an immuneinduced protease could bring new insights in these proteolytic cascades. DIMs 1, 2, 3 and 23 belong to the same family. DIMs 1, 2 and 3 are small homologous peptides containing a single disulfide bond [12] that were previously found to be strongly transcriptionally up-regulated ([27,28] and L. Troxler, personal communication). DIMs 1 and 2 possess an amidation of the C-terminus. DIM 23 is a larger molecule whose N-terminal part is similar to DIMs 1, 2 and 3. Although there is some sequence identity between DIMs 1, 2, 3 and 23, they show no similarities with any other described molecule. DIMs 4 and 14 are small peptides, which share sequence similarities and lack cysteine residues. DIM 4 has an amidation of the C-terminal amino-acid [12]. Both DIMs are lacking sequence similarity to known peptides. It is worth mentioning that in another peptidomic study, DIMs 2 and 4 were found to be expressed in the central nervous system of non-infected wandering Drosophila larvae [29]. One explanation for this finding is that in non-

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Table 1 Characteristics of DIMs DIMs Gene identity Accession Masses (Da) d a b number c DIMs corresponding or related to AMPs 9 CG10816 P36193 2401 11 CG10816 P36193 2564 15 CG10816 P36193 2767 16 CG4740 Q95NH6 2971 17 CG8175 Q24395 3026 19 CG10810 P41964 4889

Identity/homology e

Sequences f

Drosocin monosaccharide glycoform Drosocindisaccharide glycoform Drosocin trisaccharide glycoform Maturated prodomain of attacin C Metchnikowin Drosomycin

21

CG10810

P41964

5934

Drosomycin N-glycosylated

25

CG1365

P14954

4156

Cecropin A

26

CG1385

P36192

4354

Defensin

27

CG12763

P24492

9196

Diptericin O-glycosylated

GKPRPYSPRPTSHPRPIRV GKPRPYSPRPTSHPRPIRV GKPRPYSPRPTSHPRPIRV ZRPYTQPLIYYPPPPTPPRIYRA HRHQGPIFDTRPSPFNPNQPRPGPIY DCLSGRYKGPCAVWDNETCRRVCKEEGRSSGHCSPSLKCWCEGC DCLSGRYKGPCAVWDNETCRRVCKEEGRSSGHCSPSLKCWCEGC GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR amidated ATCDLLSKWNWNHTACAGHCIAKGFKGGYCNDKAVCVCRN DDMTMKPTPPPQYPLNLQGGGGGQSGDGFGFAVQGHQKVWTSDNGRHEIGLNGGYGQHLGGPYGNSEPSWKVGSTYTYRFPNF

DIMs 1 2 3 23

not related to AMPs CG18108 P82706 CG18106 O77150 CG16844 Q9V8G0 CG15066 Q9V8F5

4 14 10 12 13 24

CG15231

1666 1688 1700 9482

CG18279 CG18279 CG18279 CG18279

P82705 P83869 Q8ML70 Q8ML70 Q8ML70 Q8ML70

1722 2694 2520 2572 2650 10031

5 6 8 18

CG18279 CG18279 CG18279 CG10332

Q8ML70 Q8ML70 Q8ML70 P82701

1914 1955 2348 4625

29 30 31

CG9046 CG11129 CG31509

P13238 P06607 AAK64523

2602 3115 11936

VM26Ab fragment Yp3 fragment TotA

33

CG16712

AAF51076

6795

Protease inhibitor (Kunitz family)

GNVIINGDCRVCNVHG amidated GNVVINGDCKYCNVHG amidated GNVIINGDCRVCNVRA GNVIIGGVCQDCSPPVAENVVVGGQSYRTGRPGQGTVYINSPGAYPGALDGPIRRTGAGGGGGGGTRYPDGYSGRLPGGTYLHNKDCVGCSISGGGD GTVLIQTDNTQYIRT amidated GTQVIHAGGHTLIQTDRSQYIRKN ZLHVARPDRTVTIGNGGVYIQRS ZFHVERPGRTVDVGNGGFYIQRG ZFHVERPDRTVDFGNGGFSAQRF ZHTYDGRNGPHVFGSPGNQVYIRGQNEGTYSVPGVGGQFQNAPQRGEHVYTDEAGNTFVNRKNAGGPASHTISGPNFSAKNLGPNGAKSVGIPQRA ZFHVERPDRTVDFGNGG ZFHVERPGRTVDVGNGGF ZFHVERPDRTVDFGNGGFSAQ SRHTGPGNGSGSGAGSGNPFRSPSSQQRPLYYDAPIGKPSKTMYA ETIQLQPTQGILIPAPLAENIRVS EVNAKSPFGQRSPAHKQAAYHGMHHAQN YSDEDREADSLRIAEIIKNAQDDDSKINSTQELLDIYRRLYPSLTPEERESIDKFVNEHTDAIIIDGVPIQGGRKARIVGKIVSPGVKGLATGFFEELGSKLAQLFT LKDPICGLPAGIDGNGLIKCAAFIPSFSYHPETNSCEKFIYGGCGGNENRFGTQELCEQKCKE

a DIMs 1–24 were numbered according to the molecular masses from the mass spectra [12]. DIMs 25–33 were numbered in chronological order of HPLC detection. DIMs 7, 20, 22, 28 and 32 were deliberately omitted from the table due to a lack of sequence information, except for DIM 20, which was a doubly charged ion from DIM 24 mistakenly annotated. b CG number applied to each gene arising from complete sequencing of the Drosophila genome by Celera. c Accession numbers as described in NCBI (March 2004, www.ncbi.nlm.nih.gov). d Average of mass in Da measured from MALDI-TOF mass spectra (Fig. 1) for DIMs 1–24 or from HPLC fraction for DIMs 25–33. e Function according to Flybase (March 2004, flybase.bio.indiana.edu) for fully identified gene products or proposed function and/or homology for others. Post-translational modifications are indicated when known. f Primary structures were obtained by N-terminal and/or C-terminal sequencing. In the case of partial identification, additional residues were deduced from data bank information. When there was a mismatch between Edman sequencing and data bank information, the residue obtained by Edman sequencing is indicated in bold. Identified post-translational modification sites are underlined.

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Fig. 2. DIM 24 family precursor. All the DIMs from this family are synthesized as a unique prepropeptide. Cleavage at furin-like sites leads to the detection of DIMs 24, 12, 10 and 13 in the hemolymph and of their associated fragments.

infected flies, these molecules are produced and stocked in the central nervous system and may be released into the hemolymph after infection. DIMs 10, 12, 13 and 24 are all characterized by the presence of a pyroglutamic acid at the N-terminal extremity. Identification of their primary structure required the enzymatic removal of this post-translational modification or, in the case of DIM 13, the use of ion trap MS to obtain internal sequence information [30]. Unlike all other DIMs described in this paper, they are encoded by a single gene organized in a cluster (Fig. 2) and are synthesized as a prepropeptide containing several RRSP motifs including a dibasic furinlike cleavage site [31]. DIMs 10, 12 and 13 have strong sequence similarities. Moreover three cleavage fragments of DIMs 12 and 13 were characterized from the hemolymph after infection. DIM 6 corresponds to the N-terminal part of DIM 12, while DIMs 5 and 8 correspond to the N-terminal part of DIM 13. The function of the DIMs from this family was investigated extensively and the most interesting property identified so far was an anti-inflamatory activity. ICAM1-mediated cellular adhesion is inhibited at 58% by 10 µM DIM 13 [32]. This particular family of DIMs has been studied using either RNA interference or over-expression of the cluster, leading to the disappearance of DIMs 5, 6, 8, 10, 12, 13 and 24 or their constitutive expression, respectively. However, survival studies after infection with various microorganisms did not show any difference compared to control flies (D. Rabel, unpublished data). An additional test has been performed to investigate phagocytosis in adult fly blood cells. The phagocytosis of FITC-marked Escherichia coli was monitored, but no differences were seen between knocked out and control flies (D. Rabel, unpublished data). DIM 18 has no similarity with any known protein. It has no cysteine or amidation of the C-terminal amino-acid. DIM 31 is a peptide with no previously identified structural motifs or sequence similarity to other molecules. The gene TotA, encoding this DIM, is expressed after a septic

Fig. 3. 2D maps of silver stained proteins from hemolymph of control adult Drosophila. One hundred microgram of protein (hemolymph from about 200 flies) from control adult Drosophila were loaded on 2D gels (5–8 pH range, 17 cm immobilized pH gradient strip followed by 11% SDS-PAGE). Spot detection was performed using silver nitrate staining. This map contains around 350 spots.

injury and also following other different stresses (UV, heat, oxidative stress) [33,34].

3. Proteomic approach To complement the peptidomic studies reported above, proteins present in adult Drosophila hemolymph were investigated through 2D gel electrophoresis, which has a unique capacity for the resolution of complex mixtures of proteins (for a review see [35,36]). The optimization of sample preparation led to a well-resolved silver stained 2D map showing an average of 350 spots with molecular masses of between 15 and 150 kDa (11% SDS-PAGE), within a 5–8 pH range (Fig. 3 and [14]). Increased sample loading associated with colloidal Coomassie brilliant blue (CBB) staining afforded another reference map containing fewer spots, due to lower sensitivity of this staining (160 versus 350) but the technique was much more compatible with further MS analysis. Moreover, the use of narrow range strips significantly increased the resolution, allowing the study of proteins present at a low concentration. Recently, the 2D gel electrophoresis methodology was applied to larval Drosophila hemolymph [37,38] showing both similarities and clear differences in the protein patterns from the blood of both control Drosophila stages (third instar larvae and adult). We performed differential analyses on adult hemolymph before and 72 h after an immune challenge with the filamentous fungus Beauveria bassiana, or 6 h after inoculating the Gram-negative bacterium E. coli or the Gram-positive bacterium Micrococcus

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Table 2 Protein identification of 2D gel spots Gene identity a Proteases

Identity/homology b

1 CG16705

Prophenoloxydase activating enzyme 2 CG1102 Prophenoloxydase activating enzyme 3 CG9372 Serine protease Protease inhibitors 4 CG1857 (nec) Necrotic 5 CG6687 Necrotic-like Recognition protein 6 CG13422 GNBP-like Complement-like 7 CG10363 (TepIV) Thiolester containing protein IV protein Olfactory protein 8 CG7584 (Obp Odorant-binding protein 99c 99c) Amidation enzyme 9 CG3832 (Phm) Peptidylglycine a-hydroxylating monooxygenase Signal transduction 10 CG17919 Phosphatidylethanolamine binprotein ding protein Iron metabolism 11 CG6186 (Tsf1) Transferrin 1 entire form proteins 12 CG6186 (Tsf1) Transferrin 1 cleaved form 13 CG2216 Ferritin 1 heavy chain homolo(Fer1HCH) gue 14 CG1469 Ferritin 2 light chain homologue (Fer2LCH)

Control

Infectedc

Expression d B. bassiana ++ (6)

M. luteus =

E. coli –

i

ND

ND

++ (6) ++ (6) i i i

ND = ND = ND

ND = ND = ND

++ (7)

- - (5)

–

i

ND

ND

ND

i

–

++ (10) i - - (17)

– ND =

– ND =

- - (13)

=

=

a CG number applied to each gene arising from complete sequencing of the Drosophila genome by Celera. Fully identified gene products have the associated acronyms indicated in brackets after the CG number. b Function as described in Flybase (March 2004) for fully identified gene products or proposed function and/or homology for others. c Examples of image area showing differentially regulated spots from 2D gels of hemolymph proteins following Beauveria bassiana infection, except for CG17919 (lane 10), which is from Micrococcus luteus infected flies. d Level of expression for each spot was determined using PDQuest image analysis software: i, protein induced after the infection; ++ (x), protein with expression level increased more than fivefold after infection (variation rate is indicated in brackets); - - (x), protein with expression level decreased more than fivefold after infection (variation rate is indicated in brackets); –, protein with expression level decreased after infection with a variation rate lesser than fivefold; =, protein with expression level unchanged after infection; ND, not detected on the gel.

luteus. This study revealed that 70 of the 160 proteins detected by CBB staining were differentially expressed by at least fivefold after fungal or bacterial challenges. Regulated spots were analyzed using a classical MALDI fingerprint approach [39]. Among the differentially expressed molecules, several act in general metabolic processes and will not be described here (see [14] for details), the remainder of this review being focused on proteins more specifically related to the immune response. Recently, a similar differential strategy was used for the analysis of hemolymph proteome of Drosophila larvae [25]. A fluorescent method, where both naive and LPS-stimulated samples are labeled with different dyes, afforded the detection of 23 differentially expressed proteins. Among these, a few were detected in both studies and they will be mentioned below. 3.1. Molecules related to immune mechanisms 3.1.1. Proteases and protease inhibitors The Drosophila genome encodes 11 genes for putative prophenoloxydase activating enzymes (proPO-AEs), five of which were already found to be up-regulated on microarray studies [27]. Among these, only CG1102 and CG16705 were up-regulated upon fungal infection in our proteomic study

(Table 2 lanes 1 and 2). These molecules are serine proteases, likely to be involved in the melanization process [40]. Among 200 genes for putative serine proteases present in Drosophila genome, around 30 are zymogens, inactive protease precursors containing N-terminal disulfide-knotted motifs (CLIP domains) that are assumed to play a role in regulating the processing of proenzyme forms to active enzymes [41]. One member of this family, Psh, was identified as an essential component of the Toll pathway [8]. Another CLIP domain-containing protease, CG9372, was found to be up-regulated upon fungal infection (Table 2 lane 3) and may represent a new candidate involved in proteolytic cascades. The expression level of two serpins was altered by fungal infection. The first one corresponds to Nec (CG1857, Table 2 lane 4), which plays a key role in the protease cascade that leads to the activation of Spaetzle, the Toll ligand. Interestingly, a parallel Western blot analysis of the 2D gel using an anti-Nec antibody [7] allowed the detection of a truncated form of Nec. Moreover, a second serpin (CG6687, Table 2 lane 5), also suspected to be a truncated form, was characterized thanks to the narrow range 2D study. 3.1.2. Recognition proteins In our study, by far the most strongly induced spot following fungal infection corresponded to a GNBP-like protein,

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CG13422 (Table 2 lane 6). In Drosophila, recent genetic data have shown that two GNBPs, GNBP 1 and 3, were clearly involved in the Toll pathway at the level of Gram-positive and fungal recognition, respectively [5,9]. Our results suggest that CG13422, phylogenetically closer to GNBP 3, could be a second member of GNBP family involved in fungal recognition. 3.1.3. Complement-like proteins Other proteins clearly related to the immune response and highlighted by our proteomic study belong to a complementlike protein family called Thioester-containing proteins (TEPs). In Drosophila, six TEP-encoding genes have been identified, four of which have a thioester site perfectly conserved. Three of them, TEPs 1, 2 and 4 were transcriptionally up-regulated after an immune challenge [26–28,42]. Thanks to narrow range 2D gels a cleaved form of TEP 4 with an apparent molecular mass of 97 kDa was found to be induced after the fungal infection (Table 2 lane 7). In parallel, Vierstraete et al. [25] reported that TEP 2 is up-regulated in Drosophila larvae hemolymph after LPS stimulation. However the apparent mass of the protein on 2D gel is much lower than the calculated mass for entire TEP 2 and could indicate the truncation of this molecule in the hemolymph of larvae. 3.2. Molecules with putative roles in immune response 3.2.1. Olfactory proteins Our proteomic study identified a protein that belongs to the insect odorant-binding protein (OBPs) family, Obp 99c, as immune-responsive in adults (Table 2 lane 8). The genome of D. melanogaster exhibits 51 potential OBP genes [43]. Based on their tissue-specific expression, these proteins are thought to participate in sensing odors and/or pheromones. Recently, two molecules related to the OBP family were shown to be induced by viral and bacterial infections, respectively, pherokine-2 (Phk-2) and -3, suggesting a link between pherokines and host defense [13]. Our results support this hypothesis, showing that expression of another member of the OBP family is specifically increased after fungal infection. 3.2.2. Amidation enzyme After fungal infection in adults, peptidylglycine a-hydroxylating monooxygenase (Phm, Table 2 lane 9) was found to be induced. Phm is the rate-limiting enzyme for C-terminal a-amidation, a specific and necessary modification of numerous secretory peptides [44]. Many AMPs from different structural families share C-terminal amidation [45–49]. Several examples of the positive effects of C-terminal amidation could be found in the literature [50,51] but one of the most important role attributed to this modification is a higher stability against enzymatic proteolysis. In Drosophila, among the DIMs secreted into the hemolymph after a septic injury and described above, several occur in a

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carboxyamidated form (DIM 1, 2 and 4). However, as their function is still unknown, the role of this modification and the involvement of Phm in the immune response remain to be elucidated. 3.2.3. Signal transduction protein The CG17919 gene product, which is homologous with the mammalian phosphatidylethanolamine binding protein (PEBP), was found to be induced in hemolymph from adult flies after a Gram-positive infection (Table 2 lane 10). In LPS-stimulated larvae, a second molecule from this family, the CG18594 gene product, was found to be up-regulated. The PEBP family is a highly conserved group of proteins with homologues in a wide variety of organisms [52]. Despite their widespread expression, little is known about the role of this protein family. Various functions have been suggested for these molecules, such as binding of lipids [53], inhibition of serine proteases [54], or acting as Drosophila odorant-binding proteins [55]. Moreover, CG17919 shows homology with a mammalian molecule from the PEBP family that regulates the mitogen-activated protein kinase cascade [56] and NF-jB-dependent pathways [57]. This molecule is classified as a signal transduction protein according to Flybase. The high level of similarity between the Drosophila Toll pathway and the mammalian NF-jB signaling pathway suggests a potential role for PEBP family in the innate immune response of both invertebrates and vertebrates. 3.2.4. Iron metabolism proteins Variations in expression of two molecules involved in iron metabolism, transferrin and ferritin, were observed during our proteomic study. Transferrin (Tsf, Table 2 lane 11), a major transporter for iron in vertebrate blood, was detected as a series of post-translationally modified forms at a basal level in control 2D gels (see Fig. 3, 72 kDa, pI 6.8–7.5). All these forms were over-expressed after fungal infection. In contrast, the two subunits of the iron-storage molecule ferritin (Table 2 lane 13 and 14) were found to be downregulated in fungally-challenged adults and unchanged after bacterial infections in adults. Conversely, ferritin was found to be up-regulated in LPS-induced larvae [25]. In addition to the increase in Tsf production, fungal infection also induced proteolytic cleavage of Tsf. Eight spots corresponding to either N-terminal or C-terminal fragments were identified on 2D gels of hemolymph collected from B. bassiana infected Drosophila (see one example in Table 2 lane 12). Preliminary results indicate that this cleavage could result from a Drosophila protease and not a fungal one [14]. Two potential links between Tsf fragments and the immune response in vertebrates were recently described in the literature. Ibrahim et al. [58] reported a cationic fragment of hen ovotransferrin possessing antimicrobial activity and Stafford et al. [59,60] showed that truncated forms of Tsf induced the production of nitric oxide by LPS-stimulated goldfish mac-

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rophages. These data, in combination with the induction of Tsf fragments in Drosophila hemolymph, lead us to propose their involvement in the innate immune response, either as AMPs or as inducers. Further studies, using Drosophila mutants altered at different levels in their immune response, are underway to investigate such a hypothesis.

assistance and Dr Alain Van Dorsselaer for specific mass spectrometry facilities. This work was supported by the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer and the National Institute of Health as well as Région Alsace for financing mass spectrometry equipment.

4. Concluding remarks

References

Differential analyses after bacterial or fungal challenge showed the regulation of more than a 100 molecules in adult Drosophila hemolymph. Using differential MALDI-TOF MS, 28 peptides with a molecular mass below 15 kDa and belonging to different structural families were identified and could be classified into two groups. The first group contains the AMPs and their different isoforms. DIMs belonging to this group are likely to be effector molecules of the immune response through their antimicrobial activity. Finding of post-translational modifications such as glycosylation, N-terminal cyclisation and amidation of the C-terminus illustrate the strength of this peptidomic approach. The second group contains molecules for which the lack of similarity to any peptide prevents the proposition of any precise function. These peptides are suspected to serve as chemokines during the Drosophila immune response but the different approaches for investigating their role have so far been unsuccessful and further studies are currently being conducted. In a complementary approach using the 2D gel analyses, proteins belonging to families already linked with the Drosophila immune response were identified, such as proPO-AEs or serpins. Other molecules were highlighted, such as Obp 99c, Phm or Tsf, providing new candidates for further investigation of innate immune mechanisms. Finally, a particularly striking point was the detection of processed forms of proteins (truncated serpins or TEP 4 and transferrin fragments) after the fungal infection in adult flies. During the reviewing process of this manuscript, twoadditional proteomic analyses of the immune response of Drosophila were published: E. Vierstraete, P. Verleyen, F. Sas, G. Van den Bergh, A. De Loof, L. Arckens, L. Schoofs. The instantly released Drosophila immune proteome is infection-specific. Biochem. Biophys. Res. Commun. 317(2004)1052–1060. O. Loseva, Y. Engstrom, Analysis of signal-dependent changes in the proteome of Drosophila blood cells during an immune response, Mol. Cell. Proteomics. In press.

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