Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach

Developmental and Comparative Immunology 27 (2003) 207–215 www.elsevier.com/locate/devcompimm

Identification of immunorelevant genes from greater wax moth (Galleria mellonella ) by a subtractive hybridization approach V. Seitza,*, A. Clermontb, M. Weddec, M. Hummelb, A. Vilcinskasc, K. Schlattererd, L. Podsiadlowskie a

Max-Planck Institute Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany Institute of Pathology, Free University, Hindenburgdamm 30, 12200 Berlin, Germany c Systematic Zoology and Evolutionary Biology, University of Potsdam, Villa Liegnitz, Lenne`str. 7a, 14417 Potsdam, Germany d Institute of Clinical Chemistry and Pathobiochemistry, Free University, Hindenburgdamm 30, 12200 Berlin, Germany e Institute of Zoology, Free University, Ko¨nigin-Luise Str. 1–3, 14195 Berlin, Germany b

Received 17 July 2002; revised 12 September 2002; accepted 15 September 2002

Abstract In this study we have analyzed bacterial lipopolysaccharide (LPS) induced genes in hemocytes of the Lepidopteran species Galleria mellonella using subtractive hybridization, followed by suppressive PCR. We have found genes that show homologies to molecules, such as gloverin, peptidoglycan recognition proteins and transferrin known to be involved in immunomodulation after bacterial infection in other species. In addition, a few molecules previously not described in the innate immune reactions were detected, such as a RNA binding molecule and tyrosine hydroxylase. Furthermore, the full-length cDNA of a LPS-induced molecule with six toxin-2-like domains is described to be a promising candidate to further elucidate the relationship between toxin- and defensin-like domains in arthropod host defense. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Galleria mellonella; Lipopolysaccharide; Subtractive hybridization; Real-time PCR; Innate immunity; Gloverin; Peptidoglycan recognition protein; Toxin-2

1. Introduction Most immunological data for insects have been reported from Drosophila melanogaster [1]. In the pre-genome age immunomodulations were studied at the protein level in relatively large insects such as Abbreviations: LPS, lipopolysaccharide; PCR, polymerase chain reaction; aa, amino acids; PGRP, peptidoglycan recognition protein. * Corresponding author. Tel.: þ49-30-8413-1267; fax: þ 49-308413-1385. E-mail address: [email protected] (V. Seitz).

the Lepidopteran species Manduca sexta, Hyalophora cecropia, Bombyx mori and Galleria mellonella, as the larger body size facilitated the collection of haemolymph for protein purification [2 – 7].In G. mellonella various immunorelevant protein molecules were described [6,8 –11]. However, compared to Drosophila melanogaster only scarce molecular data are available. To fill this gap and to build a broader basis for understanding the evolution of the immune system, we have conducted a molecular approach for which no molecular pre-information is needed and which can also easily be applied to other

0145-305X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 3 0 5 X ( 0 2 ) 0 0 0 9 7 - 6

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‘non-genomic data’ organisms. We applied a wellestablished technique for immunization of Galleria first instar larvae with bacterial lipopolysaccharide (LPS) [9]. For the identification of genes differentially expressed in LPS challenged larvae, compared to naive larvae, we employed an equalizing subtractive hybridization method [12] that proved to be sufficiently sensitive to detect even small amounts of differentially expressed genes [13]. Our approach led to the detection of several differentially expressed genes, some of which have already been described as being involved in the immune reaction of other organisms. Furthermore, we report the full-length cDNA of a molecule with six toxin-2-like domains, which seems to be a good candidate for the study of phylogenetic relations of toxin- and defensin-like domains.

2. Materials and methods 2.1. Immunization G. mellonella larvae were reared in our laboratory with an artificial diet as described earlier [9]. One milligram LPS (Sigma, Taufkirchen, Germany) was dissolved in distilled water and the solution was dorsolaterally injected into last instar G. mellonella larvae [9]. Haemolymph was collected from untreated or immunized larvae after 4, 6 and 8 h. Haemolymph samples (50 ml) were then transferred into 15 ml anticoagglutinant buffer [14]. Hemocytes were then pelleted by centrifugation (10 min 4 8C, 500 g) and subsequently used for RNA-isolation. In addition whole larvae were used for mRNA isolation 3, 6 and 8 h post injection of LPS. Water was used as a control. 2.2. RNA isolation Total RNA was isolated from pooled pellet cell suspensions each containing 1 £ 107 hemocytes using the RNeasywMini-kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. From whole G. mellonella, first instar larvae total RNA was isolated using the RNeasywMaxi-kit (QIAGEN) followed by a DNase treatment (RNaseFree DNase Set; Qiagen) and repurification with

RNeasywMidi-kit (QIAGEN) according to the manufacturer’s instructions. 2.3. Subtractive hybridization Hemocyte total RNA was amplified using the SMART technique (SMARTe PCR cDNA synthesis kit, Clontech, Heidelberg, Germany). Subtractive hybridization was performed on the SMART cDNA from untreated and LPS-treated larvae (pooled mRNA from 4, 6, and 8 h post injection) using the PCR Selecte cDNA subtraction kit (Clontech) in accordance with the manufacturer’s recommendations. All PCR and hybridization steps were performed on a Perkin Elmer 9600 PCR cycler. 2.4. Screening for differential cDNAs Subtracted PCR products were cloned using the Topo T/A Cloning-kit (K4500-40; Invitrogen, Groningen, The Netherlands). A colony PCR was performed on 500 randomly picked clones employing the vector specific primers Sp6 (ATT TAG GTG ACA CTA TAG) and T7 (TAA TAC GAC TCA CTA TAG GG); cycle conditions were 96 8C 2 min; 5 £ (96 8C 15 s; 49 8C 30 s; 72 8C 1 min; ramp 45 s) and 35 £ (96 8C 15 s; 46 8C 30 s; 72 8C 1 min; ramp 45 s). One microliter of the resulting PCR products were spotted onto two nylon membranes (Roche, Lewes, UK). As an internal control to determine differences in forward- versus reverse-subtracted probe concentration an actin PCR product was dotted onto the membranes (generated with the primers actin-up (GGG ACG ATA TGG AGA AGA TCT G) and actin-low (CAC GCT CTG TGA GGA TCT TC). After UV cross-linking (UV-Stratalinker 1800; Stratagene, Amsterdam, The Netherlands) forwardsubtracted probe (using hemocyte cDNA from LPStreated larvae as tester) and reverse-subtracted probe (using hemocyte cDNA from untreated larvae as tester) were DIG-labeled (Dig High Prime Labeling and Detection Kit; Roche) and hybridized to the membranes in accordance with the users guide instructions, employing the DIG-Wash and block buffer set (Roche). Differentially expressed cDNAs were identified by much stronger hybridization signals on the membrane hybridized with a forwardsubtracted probe compared to the membrane

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hybridized with reverse-subtracted probe. The selected PCR products were analyzed on a 1% agarose gel (ethidium bromide staining) and purified with the QIAEX-2 DNA Isolation-kit (QIAGEN). The purified PCR products were sequenced in both directions on a automatic DNA sequencer (ABI 377A) employing the vector-specific primers Sp6 and T7 and were compared to our own data and to published data bases.

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2.5. RACE-PCR The full length sequence of a G. mellonella cDNA with toxin-2-like domains was obtained employing the Smart RACE cDNA amplification kit (Clontech) according to the manufacturer’s instructions, employing the gene specific primers RACE-5 (GAC ACA GAC TCC TCC ATT GAT ACC) and RACE-nested5 (CCA AAC GAA TAG AGG CGC TGT TTA C) for

Fig. 1. cDNA sequence and deduced amino acid (aa) sequences of the Gall-6tox protein (AF 394584). A 16 aa residues long signal peptide (in bold) with the cleavage site VNA/LLV (arrow) was predicted employing ‘SignalP’ (http://www.cbs.dtu.dk/services/SignalP/) which predicts signal peptides, that serve as signals for entering the secretory pathway. The six toxin-2-like domains are underlined.

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Table 1 cDNAs identified by subtractive hybridization in LPS-treated G. mellonella larvae and homologous known sequences. The Acc. Nr. of the G. mellonella sequences deposited in NCBI genebank are given as well as the Acc. Nr. of the sequences that showed the highest score in translated Blast searches against all available NCBI databases. PGRP: peptidoglycan recognition protein. Two additional identified molecules, one identical to IMPI [9] and another with homologies to insect defensins will be described in separate publications Acc. Nr.

Highest score/Acc. Nr./total length amino acids (aa)

Positives/alignment length (aa)

AF394586 AF394587 AF394583 AF394588 AF394589 AF394590 AF394591 AF394585 AF394592 AF394584 AF394584

Transferrin/A36500/681 aa PGRP/AF076481/182 aa PGRP/AB016249/196 aa Gloverin/P81048/130 aa Prophenoloxidase activating factor/AJ400904/400 aa Cobatoxin-1/AJ224689/62 aa Heat shock protein/AF315317/186 aa RNA-binding protein/L04929/135 aa Tyrosine hydroxylase/U14395/508 aa Tenascin A43902/fragment 647 aa Tenascin-C (human)/XP_005348 2201 aa

92%/129 83%/143 81%/93 63%/69 44%/132 58%/29 57%/100 87%/57 79%/171 33%/224 35%/223

the 50 -RACE PCR and the primers RACE-3 (CTT AAT ACA AGT TGT GAT CAA TCA TGT C) and RACE-nested-3 (CTT CTT TAA CGG AAT GTG TGT GGA C) for the 30 -RACE PCR. 2.6. Northern Blot An anti-sense probe (nucleotides 1 – 1231 of sequence shown in Fig. 1; Dig RNA labeling kit; Roche) was used to perform a Northern Blot analysis according to the Dig users guide for filter hybridization (Roche) with 500 ng of poly-A RNA (Oligotex mRNA Mini-kit; Qiagen) from 4, 6 and 12 h post injection whole G. mellonella first instar larvae. 2.7. Real-time PCR-cDNA quantification For exact cDNA quantification, the LightCycler rapid thermal cycler system (Roche) was used as a reliable and flexible method for quantitative RT-PCR by monitoring the continuous fluorescence of the dsDNA binding dye SYBR Green1. As a reference for relative quantification, actin was used as a house keeping gene. Furthermore, mRNA isolated from larvae injected with sterile water was used as a control to compare the effects of LPS stimulation versus wounding. cDNA synthesis: cDNA was synthesized from 1 mg of total RNA (from whole larvae), using

the GeneAmpw RNA-PCR core kit PCR (Roche) according to the instruction manual. Real-time quantitative PCR: Reactions were performed in a 20 ml volume with 0.5 mM of each primer and MgCl2 concentration optimized to 5 mM. Nucleotides, Taq DNA polymerase, and buffer were included in the LightCycler-DNA Master SYBR Green 1 mix (Roche). Primer sequences were tox-2-up: (ATT GCA TTG GAA ATT CCT GC), tox-2-low: (CAG CTT AAG GCA TTA CAT TC). The protocol consisted of a 30 s denaturation step followed by 35 cycles of 5 s denaturation at 95 8C, 7 s annealing at 58 8C, and 20 s extension at 72 8C. Detection of the fluorescence product was carried out at the end of each extension phase. The quantification data were analyzed with the LightCycler analysis software as described earlier [15]. The baseline of each reaction was equalized by calculating the mean value of the five lowest measured data points for each sample and subtracting this from each reading point. Background fluorescence was removed by setting a noise band. As a standard curve, PCR amplification was performed with cDNA template dilutions (from immunized larvae) ranging from undiluted cDNA to dilutions of 1:10 and 1:100. 3. Results A differential cDNA library was constructed from hemocytes of last instar G. mellonella larvae, which

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were either untreated or challenged with LPS. From cloned PCR products, 500 colony PCRs were performed and the resulting amplificates were spotted on several nylon membranes. Dot blots obtained from untreated or LPS treated larvae were compared after hybridization with a forward-subtracted probe (using hemocyte cDNA from LPS-treated larvae as tester) and reversesubtracted probe (using hemocyte cDNA from untreated larvae as tester), respectively. Strongly differentially expressed PCR products were sequenced and used for a BLAST (NCBI) homology search. From 20 deduced cDNAs, 12 showed homologies to known genes (Table 1). Eight of these 12 genes displayed homologies to molecules known to be involved in immunomodulation after bacterial infections such as transferrin [16], defensin-like molecules [17,18], proprophenoloxidase activating factors [19], heat shock proteins [20], gloverin [21] or peptidoglycan recognition proteins (PGRP) [22 – 27]. However, four of the 12 cDNAs showed homologies with genes that are yet to be identified as playing a direct role in the innate immune responses, namely molecules with homologies to cobatoxin-1, RNA binding molecule, tyrosine hydroxylase [28,29] and tenascin-C. The expression differences observed by dot blot analysis were confirmed by relative RT-PCR quantification, for the two PGRP sequences (Table 1) (data not shown). This study focuses on the expression of the cDNA sequence, which displays homology to tenascin-C. The RACE technology was used to obtain the fulllength Galleria molecule (Fig. 1), of 1280 bp, which

Fig. 2. A Northern Blot analysis to detect the G. mellonella molecule with toxin-2 like domains reveals a single band, comparable in size to the cDNA obtained by RACE PCR shown in Fig. 1. The number of base pairs is indicated in the left column.

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was confirmed by Northern Blot analysis (Fig. 2). Blast search with this full-length Galleria cDNA did not confirm homology to tenascin as the Galleria cDNA was significantly shorter (296 aa; 37.6 kDA) and characteristic tenascin domains as fibronectintype-3-like repeats were missing. In contrast a search for conserved domains with ‘Pfam’ (http://www. sanger.ac.uk/Software/Pfam/) suggested the presence of 6 toxin-2-like domains in our full-length cDNA (Figs. 1 and 3). To more precisely analyze the expression differences for the cDNA with toxin-2-like domains (therafter referred to as ‘Gall-6tox’; Fig. 1), an exact quantification of Gall-6tox RNA in untreated and LPS challanged larvae, was determined using a Light Cycler (Roche). PCR kinetics of actin in untreated or LPS stimulated larvae disclosed no expression differences under all conditions (Fig. 4), whereas the molecule with toxin-2-like domains was strongly upregulated after LPS stimulation. The melting temperature curves demonstrate the presence of singular specific PCR products (Fig. 4(E) and (F)). To distinguish between the effects of LPS-injection versus wounding, the larvae were treated with aqua dest without LPS. However, no upregulation was observed in this control experiment (data not shown).

4. Discussion The key feature of the innate immune system, in vertebrates as well as invertebrates, is the ability to detect and fight a broad variety of microorganisms. This is accomplished by the capacity to discriminate between self and non-self (potentially infectious) by means of molecules such as pattern recognition proteins, which contribute to the initial recognition of

Fig. 3. Alignment of the six toxin-2 like domains of the Gall-6tox protein with the ‘Pfam’ consensus toxin-2-like domain.

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Fig. 4. Relative quantification of the Gall-6tox cDNA in comparison to actin by real-time PCR. (A) PCR products of actin and Gall-6tox. (B) Logarithmic dilution series from undiluted to 1:100 show that cDNA quantification of Gall-6tox is possible. (C) Actin PCR kinetics for untreated and LPS stimulated larvae are equal. (D) Gall-6tox mRNA is upregulated upon LPS stimulation as demonstrated by the fewer cycle numbers needed to amplify Gall-6tox. (E)–(F) Melting peaks of actin (E) and Gall-6tox (F) are in accordance with agarose gel-electrophoresis (A) and show that only one specific PCR product was amplified. X-axis: cycle number in B, C and D and temperature (8C) in E and F; y-axis: fluorescence intensity.

surface structures common among invading pathogens [30]. From jawed vertebrates (Gnathostomata), animals possess (in addition to an innate immune system) an adaptive immune system which uses somatically generated antigen receptors of the B- and T-cells [31]. The innate immunity is closely linked to the adaptive immunity as it has an essential role in its activation [31]. Therefore, a better understanding of innate immunity may also elucidate the regulation processes underlying adaptive immunity. Molecules involved in innate immunity are found to be homologous in insects and vertebrates [25,26,32] giving evidence of an ancient immune system found in the ancestor of all bilaterian animals or even all metazoa. To identify genes involved in the innate immune reaction of G. mellonella, we performed an equalizing

hybridization method followed by suppressive PCR [12,13] employing hemocytes from untreated and LPS-treated G. mellonella larvae. From 20 differential cDNAs analyzed, 12 showed homologies to molecules in the NCBI databases (Table 1) eight of which had known immunorelevant functions. For example one putative gene product showed homology to a prophenoloxidase activating factor which is associated with the prophenoloxidase activating system, involved in non-self recognition and defense responses in invertebrates [19]. Another cDNA displayed homology to transferrin, which is well known for its role in removing iron ions in vertebrates thereby creating unfavorable environments for bacteria [16] and might also be involved in an antibacterial iron-withholding strategy in insects [33,

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34]. Further canditates which were found to be differentially expressed in LPS-treated larvae displayed homology to the family of the PGRP which is described to play a role in the innate immunity of insects and vertebrates [25]. Four of the twelve cDNAs represent molecules which were not yet considered as relevant for innate or adaptive immune reaction. The fact that these genes have not yet been described in the context of an immune reaction might be due to an enrichment of rare RNAs by our technical approach [12,13]. Three of these sequences showed in NCBI Blast searches homologies to the RNA-binding protein (RBP1) [28], the tyrosine hydroxylase [29] and cobatoxin-1 [35], respectively. The cDNA with homology to cobatoxin-1 might encode for a new Galleria defensin-type molecule, as domain analysis with ‘Pfam’ algorithm suggests the presence of an Arthro-defensin domain. We focused on a G. mellonella sequence that initially showed weak homology to vertebrate tenascins (Table 1). However, the full-length cDNA (Fig. 1) obtained by RACE-PCR was much shorter than tenascin molecules and furthermore characteristic tenascin domains (e.g. fibronectin type-3-like repeats) were missing making a relation to tenascins unlikely. Moreover, thorough analysis of this cDNA with ‘Pfam’ revealed the presence of six toxin-2-like domains (Fig. 3), a domain known from scorpion short toxins. Scorpion short toxins (30 –40 residues) are a family of peptides in scorpion venoms, toxic to mammals, insects and crustaceans [36,37]. Previous studies have shown that irrespective of their size, sequence and function, scorpion toxins and also all insect defensins use a similar folding pattern namely the cystein-stabilized ab(Csab) motif [38,39]. This involves an a-helix including an invariant sequence (C – X –X – X – C) linked by two disulfide bridges to the last strand of a bstructure which includes another invariant sequence (C –X – C) [40]. Based on these structural similarities it was speculated that insect defensins and scorpion toxins could have a common ancestor with a similar structural organization [38,41]. In scorpions, additionally to toxins, several cystein-rich antibacterial peptides have been isolated from the hemolymph [42] and there is an evidence for insect defensin-like peptides in scorpion venom [38].

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Phylogenetic analysis indicates that these molecules belong to defensins [38,43]. Taken together defensins can be found in the hemolymph and venom of scorpions and the hemolymph of insects, whereas toxins acting on ionic channels, are a feature of scorpions and not insects. Under a phylogenetic perspective this lead us to a model in which first defensin-like antimicrobial molecules were present in arthropod phylogeny and toxins evolved from these molecules as an apomorphy of scorpions in their venom glands. We conclude that our Gall-6tox RNA, most likely encodes an antimicrobial protein, as it was strongly induced upon LPS-treatment in G. mellonella larvae (Fig. 4). The 6 toxin-2 like domains suggests it to be a promising candidate to further elucidate the relationship and evolution of toxin-like and defensin-like domains.

Acknowledgements We thank L. Udvarhelyi for his editorial help.

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