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Induction of a putative serine protease transcript in immune challenged Drosophila
Pergamon
Developmental and Comparative Immunology, Vol. 20, No. 4, pp. 265~272, 1996 Copyright 0 1996 Published by Elsevier Science Ltd. All rights resemd printedin Great Britain 014MOw96 $15.00+0.00
PIE SO145-305X(96)00016-X
INDUCTION
OF A PUTATIVE SERINE PROTEASE TRANSCRIPT IMMUNE CHALLENGED Drosophila
IN
Christine Coustau,* Thomas Rocheleau,* Yves Carton,t Anthony J. Nappi* and Richard H. ffrench-Constant* *Department of Entomology, University of Wisconsin, Madison, WI 53706, U.S.A. tLaboratoire Populations, GBrktique et Evolution, CNRS, 91198 Gif suryvette, France *Department of Biology, Loyola University Chicago, Chicago, IL 60626, U.S.A.
(Submitted December 1995; Accepted April 1996)
qAbstract-In an effort to identify serine proteases involved in the insect’s immune response, we used a degenerate PCR approach to amplify putative serine protease gene fragments in Drosophila. Sequencing of the cloned PCR products identified one serine protease previously isolated in D. melunogaster (SERl/ SERZ), as well as two novel putative serine protease gene fragments (SP2, SP3). The involvement of the corresponding genes in the immune response was examined by analyzing their expression in larval mRNA following both parasitic and bacterial exposures. The overexpression of one of the serine proteasesrelated mRNAs in immune challenged larvae suggests its involvement in the Drosophila immune response. Copyright Q 1996 Published by Elsevier Science Ltd.
qKeywords-Serine proteases; Immune response; Drosophila; Leptopilina; Parasitoid infection; Bacterial injection.
Introduction Together with the production of antibacterial peptides, the activation of the phenoloxidase (PO) system is an impor-
Address correspondence to Christine Coustau, Centre de Biologie et d’Ecologie, UMR 5555 du CNRS, Ave de Villeneuve, 66860 Perpignan cedex, France. 265
tant component of the biochemical reby sponse of insects to infection prokaryotic or eukaryotic organisms (l4). Insect phenoloxidases catalyze key steps in the formation of eumelanin and sclerotin, dark pigments accumulating around wounds or encapsulated parasites. The enzyme is present as an inert zymogen (prophenoloxidases: proP0) and is activated in a cascade of reactions reminiscent of the system that activates blood clotting in mammals (5). Studies on various insect species suggest that serine proteases are involved in the activation of prop0 (6-9). In Drosophila melanogaster, the prop0 system contains four proenzymes activated by serine proteases, one of them at least being of trypsin type (lO12). Although the melanization pathway is well characterized (4), none of the serine proteases involved in prophenoloxidase activation have yet been identified at the molecular level. Set-me proteases represent a very large enzyme family involved in a wide variety of physiological processes including digestion, the processing of hormones and proteins, developmental regulation, blood coagulation and the complement cascades (5,13). In insects, these enzymes have mainly been studied in hematophagous ectoparasites because of their potential use as vaccine antigens (14-17), but they remain largely uncharacterized.
266
C. Coustau et al.
In order to identify the serine proteases involved in the immune responses of the model insect Drosophila melanogaster, we used degenerate PCR to amplify serine protease gene fragments. Two D. melanogaster strains showing different immune capabilities against the parasitoid wasp Leptopilina boulardi were used to examine the involvement of putative serine proteases in the insect immune response. Larvae from the immune reactive or resistant (R) strain recognize and encapsulate eggs of the parasite within 24 h, whereas larvae from the susceptible (S) strain do not exhibit a melanotic encapsulation response (18). Expression of putative serine proteases was examined in larvae from these two strains following either parasitoid or bacterial exposure. Using this approach, we report the isolation of two novel putative serine protease gene fragments, one of which appears to be involved in the Drosophila immune response.
frozen in liquid nitrogen. Unparasitized control larvae were collected simultaneously. One to two hundred larvae from each parasitized strain were kept for an additional 7 days and the percentage of parasitization assessed (presence of melanotic capsules in pupae of the Rstrain or developing wasp larvae in pupae of the S-strain). Only samples exhibiting at least 85% infection were used to prepare RNA samples. Bacterial exposure was initiated by piercing the larvae with a fine needle (0.4 mm) previously dipped in a culture of E. co/i. This stimulation has been previously shown to induce production of a strong antibacterial response in both R and S fly strains (20). Larvae exposed to bacteria were frozen in liquid nitrogen at 24 h post-exposure, alongside unexposed control larvae.
Northern
Materials and Methods Drosophila Strains The origin of the D. melanogaster and L. boulardi parasitoid strains, as well as details of parasitoid rearing, have been described previously (19). Two Drosophila strains were used. One resistant line (Rstrain 940) which is highly immune reactive to the parasitoid wasp L. boulardi, and a second susceptible strain (S-strain 22) which is highly susceptible to parasitoid attack.
Infection
Experiments
Drosophila larvae (54 f 4 h old) were exposed to ovidepositing female L. boulardi for 4 h. Larvae were collected at 24 h post-exposure and immediately
Blot Analysis
Total RNA from control and treated Sand R-strains of Drosophila was extracted and poly(A+) RNA was purified on oligo-dT-cellulose columns (messenger RNA isolation kit, Stratagene) according to the manufacturer’s instructions. RNA samples (3 pg) were separated on denaturing (formaldehyde) 1% agarose gels, capillary blotted to a nylon membrane (Hybond-N, Amersham) and cross-linked by UV irradiation. RNA blots were prehybridized for 4 h at 42°C (50% formamide, 5 x SSPE, 5 x Denhardt’s solution, 10% dextran sulfate, 0.5% SDS and 250 pg/mL denatured salmon sperm DNA), and hybridized over night at 42°C with 32P-labeled probes. Filters were then washed four times for 30 min (0.2 x SSPE +0.5% SDS at 65°C) and exposed to Xray film at -80°C with an intensifying screen. The integrity and concentration of loaded RNA samples were confirmed by hybridizing all blots to the standard ribosomal cDNA rp49.
Serine protease transcript in
Drosophila
267
PCR
Results and Discussion
Genomic DNA was extracted from the Drosophila standard laboratory strain Oregon-R (20), and PCR amplification was performed using two previously described degenerate primers (21). These primers are based on the highly conserved regions surrounding the His-57 and the Ser-195 residues common to all serine proteases (bovine chymotrypsin numbering; 22). PCR was performed for 40 cycles with 2 min denaturation at 94”C, 1 min annealing at WC, and 2 min extention at 72°C after a single 2 rain period of denaturation at 94°C. Concentrations of 2, 3.5 and 5 mA4 of MgClz were tested for this reaction.
Ampkjkation of Putative Serine Protease Gene Fragments
Molecular Cloning and DNA Sequencing PCR products were purified by agarose gel electrophoresis, ligated into the EcoRI site of the pCRTMII vector (Invitrogen) and cloned (TA CloningTM Kit, Invitrogen) according to the manufacturer’s instructions. DNA minipreparations of positive recombinant clones were then digested with EcoRI and run on a 1% agarose gel to assess the sizes of the inserts. DNA sequencing of selected clones was carried out by the dideoxy chain termination method using the Sequenase II kit (U.S. Biochemical).
DNA Labeling DNA fragments of putative serine protease genes were purified from low melting point agarose (SeaPlaque@, FMC@) and labeled with 32P by random priming (random primed DNA labeling kit, Boehringer-Manheim). Probes were purified using Biospina chromatography columns (Bio-Rad) and used at a concentration of lo6 cpm/mL of hybridizing solution.
Three major bands of PCR products approximately 440, 500 and 600 bp in length were detected (Fig. 1). Since the distance separating the relative His-57 and Ser-195 residues ranges from 390 bp to 490 bp in most of the serine proteases identified in insects so far (21,23,24), we selected PCR products of approximately this size for cloning in the pCRI1 vector (Invitrogen). Three clones of the predicted size showed high homology with serine proteases. The predicted amino acid sequences of these PCR fragments are shown in Figure 2. The insert from clone SP# 1 showed a 100% identity with the corresponding fragments of the serine proteases genes SERl and SER2 characterized in D. melanogaster by Yun and Davis (24) (sequence not shown). The sequences from clones SP# 2 and SP# 3 do not correspond to any serine proteases previously identified in D. melanogaster. However, the region surrounding the Asp-102 residue, which, together with the His-57 and the Ser-195, comprises the catalytic triad characteristic of these enzymes (22), is highly conserved with other serine proteases. Comparison of the deduced amino-acid sequence of SP# 2 and SP# 3 with sequences found in GENBANK using the program “Blast” showed strong similarities with, the Drosophila Easter precursor (45% identity, 65.5% similarity), and a Lucilia cuprina serine protease (59O/ identity, 73% similarity) (Fig. 2). The product of the Easter gene is an extracytoplasmic serine protease belonging to the trypsin family, that is required for establishment of the embryo dorsal-ventral pattern (24). The L. cuprina serine protease, although partially characterized, also appears to be a trypsin-like enzyme (“sbtrf” in Ref. 21). The similarity of SP2 and SP3 with these insect serine proteases and other trypsin-
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Figure 1. Agarose gel of amplification productsfrom PCR using degenerate serine proteases primers, at a range of MgCIP concentrations. The arrow indicates the band purified for further cloning. S = 1 kb ladder.
poly(A+) RNA from both control and treated larvae of the S- and R-strains (Fig. 3). The additional signal detected at 3.5 kb (Fig. 3) is consistent with the results of Yun and Davis (24) on SERl and SER2 expression, Although lower levels of SPI-related mRNA were detected in control and parasitoid infected Expression of Putative Serine larvae from the R-strain as compared Pro teases in Immune Challenged with other samples, these differences in Larvae expression are not correlated with a The low levels of SPZrelated mRNA in particular treatment and do not suggest control larvae from S- and R-strains were an involvement of SPl in the immune increased significantly after infection by response. The high expression of SERl/ L. boulardi (Fig. 3). The stimulation of SER2 in larval gut tissues led Yun and expression by the parasite, and the even Davis (24) to suggest that a major function of the gene products may be to stronger stimulation of SPZrelated aid in digestion. mRNA by bacterial infection, implicates When SP3 PCR products were used as the SPZrelated gene in the Drosophila probe on the same RNA blot, no signal immune response. High levels of SPl (SERl, SER2)- was observed, even after prolonged exrelated genes were detected at 1.0 kb in posure. like molecules (complete blast search not shown) suggests that the gene fragments we have identified correspond to serine proteases of the trypsin family.
Serine protease transcript in Drosophila
269
* SP#2 EAST SP#2 EAST SP#2 EAST SP#2 EAST
SP#3 LCU
WVVTAAHCIHTM.. ..GRNLTAAILGEWNRDTDPDCENDLNGVRECAPPH : : * I * : . IIII: :l:II.II::.. :l:IlIi 1:.1:::11111 YVITASHCVNGKALPTDWRLSGVRLGEWDTNTNPDCEVDVRGMKDCAPPH * IRVTIDRILPHAQY..SELNYRNDIALLRLSRPVNWLQMQNLEPVCLPPQ : I:lIl IlIlllIl...l:: :: . : : l.:Il::I . . I ~DVPVERTIPH~DYIPASKNQVNDIALLRLAQQVEYTDF..VRPICLPLD RGRYANQLAGSAADVSGWGKTESSGSSKIKQKAMLHIQPQDQCQEAFYKD . . . :. I. II.IIIIll :.I.:) II :. . I:lI:.: VNLRSATFDGITMDVAGWGKTEQLSASNLKLKAAVEGSRMDECQNVYSSQ *
:
.:
TKITLADSQMCAGGEIGVDSCSGDSGGP. .I I.I.IIIIII. lllll.IIIIII .DILLEDTQMCAGGKEGVDSCRGDSGGPL
* WWTAAHCLDTPTTVSNLRIRAGSNKRTYGGVLVEVAAIKAHEAYNSNSK IIIIIIIII:...I I I: IIII. :III:I.III:I.II:II.... WVVTAAHCLQSVST SVLKARAGSSYWNSGGVWSVAAFKNHEGYNPKTM l
SP#3 LCU
INDIGWRLKTKLTFGSTIKAITMASATPAHGSAASISGWDKTSTDGPSS :lIl:l:lI...lI::IlllII.:..l.lI:I.II.:III:.II.:l. VNDIAVIRLSSSLTMSSTIKAIALTTAAPANGAAATVSGWGTTSSGGSIP
. *
SP#3 LCU
ATLLFVDTRIVGRSQCGSSTYGYGSFIKATMICAAATNKDACQGDSGGPL Il:.IIII . ..lI.lIIIIIIIl I I : I I :llll.ll:llllllll AQLRYVDLKIVGRTQCASSTYGYGSQIKPSMICAYTVGKDSCQGDSGGPL
Figure 2 Alignment of the deduced amino acid sequences of the 2 novel putative serine proteases gene fragments amplified from D. melanogaster (SP# 2, SP# 3) with the respective serine proteases corresponding to the highest ‘blast scores’. Identical residues are indicated by (I), and conserved residues by (:). EAST_DROM: D. melanogaster serine protease Easter precursor (23); LCUO76931:serine proteinase Lucilia cuprina (sbtrf in 21). Asterisks indicate the His-57, Asp-102 and Ser-195 residues characteristic of serine proteases.
by L.
Involvement of SPZ-related Gene in the Immune Response
boulardi, the expression of antibacterial
The stimulation of expression of SP2related gene in both S- and R-strain hosts following parasitic infection and bacterial exposure strongly suggests its involvement in the immune response, but raises the question of the apparent lack of specificity of this response. Although bacterial exposure elicited a significantly stronger induction of SP2 than did the parasite infection, we cannot specifically relate SP2 gene induction to the antibacterial response. Our previous studies
transcripts (cecropin and diptericin) is strongly induced in the S-strain, but not in the R-strain (20). Therefore, SP2 and antibacterial transcripts are not induced in a similar way. The induction of SP2 also does not seem to be directly correlated with phenoloxidase activity, since a strong increase in both monophenoloxidase and diphenoloxidase has been observed in hemolymph from the Rstrain 24 h after infection, but not in the hemolymph from the S-strain (25). Therefore, SP2 could participate in a reaction
showed that 24 h after infection
270
C. Coustau et al.
S. strain
R. strain
c12c12
SP #l
SP #2
Figure 3. Leptopilina
Expression of SP# 1, SP# 2 and SP# 3 related gene transcripts in mRNA of control (C), boulardi-infected (1) and E. co/i-exposed (2) larvae from Drosophila S and R strains.
occurring systematically after stimulation of the internal defence system, independently of the type of stimulation and of the relative success or failure of the infection. Although the involvement of serine proteases in the insect immune response has only been linked with the activation of the PO system during melanotic reactions, there is a considerable potential for these enzymes also to participate immunologically in further pathways.
The extensive body of work carried out on mammalian immunogenetics reveals that serine proteases are involved in the regulation of diverse key reactions of the inflammation and acute phase responses taking place shortly after trauma or infections (26-28). Knowing that serine proteases are an evolutionary old and well-conserved enzyme family (5,29,30), and that they also represent a large gene family in insects (21), it seems likely that
Serine protease transcript in Drosophila
271
these enzymes may participate in a diversity of insects’ immune reactions. At present it is unclear as to exactly how SP2 is employed in the immune system of D. melanogaster, but its overexpression in immune challenged larvae suggests it may play a key role in this insect’s immune response.
Acknowledgements-We thank F. Shotkoski for his skilled assistance in the laboratory, S. Paskewitz for provision of the serine protease degenerate primers and critical reading of the manuscript, and T. Yoshino for comments on the manuscript. A. J. N. acknowledges support from the National Science Foundation (IBN 950 4796)
References 1. Boman, H. G. Antibacterial peptides: key components needed in immunity. Cell 65:205207; 1991. 2. Kimbrell, D. A. Insect antibacterial proteins: not just for insects and against bacteria. BioEssays 1312657-663; 1991. 3. Hultmark, D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 95:178-184; 1993. 4. Nappi, A. J.; Vass, E. Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res. 6:117-126; 1993. 5. Neurath, H. Evolution of proteolytic enzymes. Science 224:35&357; 1984. 6. Dohke, K. Studies on prophenoloxidaseactivating enzyme from cuticle of the silkworm Bombyx mori. I. Activation reaction by the enzyme. Archs Biochem. Biophys. 157:203-209; 1973. 7. Sugumaran, M.; Saul, S. J.; Ramesh, N. Endogenous protease inhibitors prevent undesired activation of prophenoloxidase in insect hemolymph. Biochem. Biophys. Res. Comnmn. 13211241129; 1985. 8. Yoshida, H.; Ashida, M. Microbial activation of two serine enzymes and prophenoloxidase in the plasma fraction of hemolymph of the silkworm, Bombyx mori. Insect. Biochem. 163:539-545; 1986. 9. Ashida, M.; Kinoshita, K.; Brey, P. T. Studies on prophenoloxidase activation in the mosquito Aedes aegypti L. Eur. J. B&hem. 188:507-515; 1990. 10. Seybold, W. D.; Meltmr, P. S.; Mitchell, H. K. Phenol oxidase activation in Drosophila: a cascade of reactions. Biochem. Genet. 13:85108; 1975. 11. Rixki, T. M.; Rixki, R. M.; Bellotti, R. A. Genetics of a Drosophila phenoloxidase. Mol. Gen. Genet. 201:7-13; 1985. 12. Yonemura, M.; Kasatani, K.; Asada, N.; Oh&hi, E. Involvement of a serine protease in the activation of prophenoloxidase in Drosophila melanogaster. Zool. Sci. (Tokyo) 8:865-867; 1991. 13. Neurath, H. The versatility of proteolytic enzymes. J. Cell. Biochem. 32:35-49; 1986.
14. Baron, R. W.; Colwell, D. D. Mammalian immune responses to myasis. Parasitol. Today 7:353-355; 1991. 15. Elvin, C. M.; Whan, V.; Riddles, P. W. A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua). Mol. Gen. Genet. 240:132-139; 1993. 16. Casu, R. E.; Jarmey, J. M.; Elvin, C. M.; Eisemann, C. H. Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina. Insect Mol. Biol. 33:159-170; 1994. 17. Casu, R. E.; Pearson, R. D.; Jarmey, J. M.; Cadogan, L. C.; Riding, G. A.; Tellam, R. L. Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA. Insect Mol. Biol. 34:201-211; 1994. 18. Carton, Y.; Frey, F.; Nappi, A. Genetic determinism of the cellular immune reaction in Drosophila melanogaster. Heredity 69:393-399; 1992. 19. Carton, Y.; Nappi, A. The Drosophila immune reaction and the parasitoid capacity to evade it: genetic and coevolutionary aspects. Acta Ecol. 121389104; 1991. 20. Coustau, C.; Carton, Y.; Nappi, A. J.; Shotkoski, F. A. S.; ffrench-Constant, R. H. Differential induction of antibacterial transcripts in Drosophila susceptible and refractory to parasitism by Leptopilina boulardi. Insect Mol. Biol. 3:167-172; 1996. 21. Elvin, C. M.; Vuocolo, T.; Smith, W. J. M.; Eisemann, C. H.; Riddles, P. W. An estimate of the number of serine protease genes expressed in sheep blowtly larvae (Lucilia cuprina). Insect Mol. Biol. 32:105-115; 1994. 22. Brown, J. R.; Hartley, B. S. Location of disulphide bridges by diagonal paper electrophoresis. The disulphide bridges of bovine chymotrypsinogen. A. Biochem. J. 101:214228; 1966. 23. Chasan, R.; Anderson, K. V. The role of Easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo. Cell 56z391-400; 1989.
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24. Yun, Y.; Davis, R. L. Levels of RNA from a family of putative serine protease genes are reduced in Drosophila nzelanogaster dunce mutants and are regulated by cyclic AMP. Mol. Cell. Biol. 92:692-700; 1989. 25. Nappi, A.; Carton, Y.; Frey, F. Parasiteinduced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster. Arch. Ins. Biochem. Biophys. 18:159-168; 1991. 26. Bamnann, H.; Gauldie, J. The acute phase response. Immunol. Today 152:74-80; 1994. 21. Vyse, T. J.; Bates, G. P.; Walport, M. J.; Morley, B. J. The organization of the human complement factor I gene (IF): a member of the
C. Coustau et al.
serine protease gene family. Genomics 2490-98; 1994. 28. Thia, K. Y. T.; Jenkins, N. A.; Gilbert, D. J.; Copeland, N. G.; Smyth, M. J. The natural killer cell serine protease gene Lmet 1 maps to mouse chromosome 10. Immunogenetics 41:4749; 1995. 29. Kraut, J. Serine proteases: structure and mechanism of catalysis. Ann. Rev. Biochem. 46:331-358; 1977. 30. Sakanari, J. A.; Staunton, C. E.; Eakin, A. E.; Craik, C. S.; McKerrow, J. H. Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. Proc. Natl. Acad. Sci. U.S.A. 8648634867; 1989.
Developmental and Comparative Immunology, Vol. 20, No. 4, pp. 265~272, 1996 Copyright 0 1996 Published by Elsevier Science Ltd. All rights resemd printedin Great Britain 014MOw96 $15.00+0.00
PIE SO145-305X(96)00016-X
INDUCTION
OF A PUTATIVE SERINE PROTEASE TRANSCRIPT IMMUNE CHALLENGED Drosophila
IN
Christine Coustau,* Thomas Rocheleau,* Yves Carton,t Anthony J. Nappi* and Richard H. ffrench-Constant* *Department of Entomology, University of Wisconsin, Madison, WI 53706, U.S.A. tLaboratoire Populations, GBrktique et Evolution, CNRS, 91198 Gif suryvette, France *Department of Biology, Loyola University Chicago, Chicago, IL 60626, U.S.A.
(Submitted December 1995; Accepted April 1996)
qAbstract-In an effort to identify serine proteases involved in the insect’s immune response, we used a degenerate PCR approach to amplify putative serine protease gene fragments in Drosophila. Sequencing of the cloned PCR products identified one serine protease previously isolated in D. melunogaster (SERl/ SERZ), as well as two novel putative serine protease gene fragments (SP2, SP3). The involvement of the corresponding genes in the immune response was examined by analyzing their expression in larval mRNA following both parasitic and bacterial exposures. The overexpression of one of the serine proteasesrelated mRNAs in immune challenged larvae suggests its involvement in the Drosophila immune response. Copyright Q 1996 Published by Elsevier Science Ltd.
qKeywords-Serine proteases; Immune response; Drosophila; Leptopilina; Parasitoid infection; Bacterial injection.
Introduction Together with the production of antibacterial peptides, the activation of the phenoloxidase (PO) system is an impor-
Address correspondence to Christine Coustau, Centre de Biologie et d’Ecologie, UMR 5555 du CNRS, Ave de Villeneuve, 66860 Perpignan cedex, France. 265
tant component of the biochemical reby sponse of insects to infection prokaryotic or eukaryotic organisms (l4). Insect phenoloxidases catalyze key steps in the formation of eumelanin and sclerotin, dark pigments accumulating around wounds or encapsulated parasites. The enzyme is present as an inert zymogen (prophenoloxidases: proP0) and is activated in a cascade of reactions reminiscent of the system that activates blood clotting in mammals (5). Studies on various insect species suggest that serine proteases are involved in the activation of prop0 (6-9). In Drosophila melanogaster, the prop0 system contains four proenzymes activated by serine proteases, one of them at least being of trypsin type (lO12). Although the melanization pathway is well characterized (4), none of the serine proteases involved in prophenoloxidase activation have yet been identified at the molecular level. Set-me proteases represent a very large enzyme family involved in a wide variety of physiological processes including digestion, the processing of hormones and proteins, developmental regulation, blood coagulation and the complement cascades (5,13). In insects, these enzymes have mainly been studied in hematophagous ectoparasites because of their potential use as vaccine antigens (14-17), but they remain largely uncharacterized.
266
C. Coustau et al.
In order to identify the serine proteases involved in the immune responses of the model insect Drosophila melanogaster, we used degenerate PCR to amplify serine protease gene fragments. Two D. melanogaster strains showing different immune capabilities against the parasitoid wasp Leptopilina boulardi were used to examine the involvement of putative serine proteases in the insect immune response. Larvae from the immune reactive or resistant (R) strain recognize and encapsulate eggs of the parasite within 24 h, whereas larvae from the susceptible (S) strain do not exhibit a melanotic encapsulation response (18). Expression of putative serine proteases was examined in larvae from these two strains following either parasitoid or bacterial exposure. Using this approach, we report the isolation of two novel putative serine protease gene fragments, one of which appears to be involved in the Drosophila immune response.
frozen in liquid nitrogen. Unparasitized control larvae were collected simultaneously. One to two hundred larvae from each parasitized strain were kept for an additional 7 days and the percentage of parasitization assessed (presence of melanotic capsules in pupae of the Rstrain or developing wasp larvae in pupae of the S-strain). Only samples exhibiting at least 85% infection were used to prepare RNA samples. Bacterial exposure was initiated by piercing the larvae with a fine needle (0.4 mm) previously dipped in a culture of E. co/i. This stimulation has been previously shown to induce production of a strong antibacterial response in both R and S fly strains (20). Larvae exposed to bacteria were frozen in liquid nitrogen at 24 h post-exposure, alongside unexposed control larvae.
Northern
Materials and Methods Drosophila Strains The origin of the D. melanogaster and L. boulardi parasitoid strains, as well as details of parasitoid rearing, have been described previously (19). Two Drosophila strains were used. One resistant line (Rstrain 940) which is highly immune reactive to the parasitoid wasp L. boulardi, and a second susceptible strain (S-strain 22) which is highly susceptible to parasitoid attack.
Infection
Experiments
Drosophila larvae (54 f 4 h old) were exposed to ovidepositing female L. boulardi for 4 h. Larvae were collected at 24 h post-exposure and immediately
Blot Analysis
Total RNA from control and treated Sand R-strains of Drosophila was extracted and poly(A+) RNA was purified on oligo-dT-cellulose columns (messenger RNA isolation kit, Stratagene) according to the manufacturer’s instructions. RNA samples (3 pg) were separated on denaturing (formaldehyde) 1% agarose gels, capillary blotted to a nylon membrane (Hybond-N, Amersham) and cross-linked by UV irradiation. RNA blots were prehybridized for 4 h at 42°C (50% formamide, 5 x SSPE, 5 x Denhardt’s solution, 10% dextran sulfate, 0.5% SDS and 250 pg/mL denatured salmon sperm DNA), and hybridized over night at 42°C with 32P-labeled probes. Filters were then washed four times for 30 min (0.2 x SSPE +0.5% SDS at 65°C) and exposed to Xray film at -80°C with an intensifying screen. The integrity and concentration of loaded RNA samples were confirmed by hybridizing all blots to the standard ribosomal cDNA rp49.
Serine protease transcript in
Drosophila
267
PCR
Results and Discussion
Genomic DNA was extracted from the Drosophila standard laboratory strain Oregon-R (20), and PCR amplification was performed using two previously described degenerate primers (21). These primers are based on the highly conserved regions surrounding the His-57 and the Ser-195 residues common to all serine proteases (bovine chymotrypsin numbering; 22). PCR was performed for 40 cycles with 2 min denaturation at 94”C, 1 min annealing at WC, and 2 min extention at 72°C after a single 2 rain period of denaturation at 94°C. Concentrations of 2, 3.5 and 5 mA4 of MgClz were tested for this reaction.
Ampkjkation of Putative Serine Protease Gene Fragments
Molecular Cloning and DNA Sequencing PCR products were purified by agarose gel electrophoresis, ligated into the EcoRI site of the pCRTMII vector (Invitrogen) and cloned (TA CloningTM Kit, Invitrogen) according to the manufacturer’s instructions. DNA minipreparations of positive recombinant clones were then digested with EcoRI and run on a 1% agarose gel to assess the sizes of the inserts. DNA sequencing of selected clones was carried out by the dideoxy chain termination method using the Sequenase II kit (U.S. Biochemical).
DNA Labeling DNA fragments of putative serine protease genes were purified from low melting point agarose (SeaPlaque@, FMC@) and labeled with 32P by random priming (random primed DNA labeling kit, Boehringer-Manheim). Probes were purified using Biospina chromatography columns (Bio-Rad) and used at a concentration of lo6 cpm/mL of hybridizing solution.
Three major bands of PCR products approximately 440, 500 and 600 bp in length were detected (Fig. 1). Since the distance separating the relative His-57 and Ser-195 residues ranges from 390 bp to 490 bp in most of the serine proteases identified in insects so far (21,23,24), we selected PCR products of approximately this size for cloning in the pCRI1 vector (Invitrogen). Three clones of the predicted size showed high homology with serine proteases. The predicted amino acid sequences of these PCR fragments are shown in Figure 2. The insert from clone SP# 1 showed a 100% identity with the corresponding fragments of the serine proteases genes SERl and SER2 characterized in D. melanogaster by Yun and Davis (24) (sequence not shown). The sequences from clones SP# 2 and SP# 3 do not correspond to any serine proteases previously identified in D. melanogaster. However, the region surrounding the Asp-102 residue, which, together with the His-57 and the Ser-195, comprises the catalytic triad characteristic of these enzymes (22), is highly conserved with other serine proteases. Comparison of the deduced amino-acid sequence of SP# 2 and SP# 3 with sequences found in GENBANK using the program “Blast” showed strong similarities with, the Drosophila Easter precursor (45% identity, 65.5% similarity), and a Lucilia cuprina serine protease (59O/ identity, 73% similarity) (Fig. 2). The product of the Easter gene is an extracytoplasmic serine protease belonging to the trypsin family, that is required for establishment of the embryo dorsal-ventral pattern (24). The L. cuprina serine protease, although partially characterized, also appears to be a trypsin-like enzyme (“sbtrf” in Ref. 21). The similarity of SP2 and SP3 with these insect serine proteases and other trypsin-
268
C. Coustau et al.
Figure 1. Agarose gel of amplification productsfrom PCR using degenerate serine proteases primers, at a range of MgCIP concentrations. The arrow indicates the band purified for further cloning. S = 1 kb ladder.
poly(A+) RNA from both control and treated larvae of the S- and R-strains (Fig. 3). The additional signal detected at 3.5 kb (Fig. 3) is consistent with the results of Yun and Davis (24) on SERl and SER2 expression, Although lower levels of SPI-related mRNA were detected in control and parasitoid infected Expression of Putative Serine larvae from the R-strain as compared Pro teases in Immune Challenged with other samples, these differences in Larvae expression are not correlated with a The low levels of SPZrelated mRNA in particular treatment and do not suggest control larvae from S- and R-strains were an involvement of SPl in the immune increased significantly after infection by response. The high expression of SERl/ L. boulardi (Fig. 3). The stimulation of SER2 in larval gut tissues led Yun and expression by the parasite, and the even Davis (24) to suggest that a major function of the gene products may be to stronger stimulation of SPZrelated aid in digestion. mRNA by bacterial infection, implicates When SP3 PCR products were used as the SPZrelated gene in the Drosophila probe on the same RNA blot, no signal immune response. High levels of SPl (SERl, SER2)- was observed, even after prolonged exrelated genes were detected at 1.0 kb in posure. like molecules (complete blast search not shown) suggests that the gene fragments we have identified correspond to serine proteases of the trypsin family.
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* SP#2 EAST SP#2 EAST SP#2 EAST SP#2 EAST
SP#3 LCU
WVVTAAHCIHTM.. ..GRNLTAAILGEWNRDTDPDCENDLNGVRECAPPH : : * I * : . IIII: :l:II.II::.. :l:IlIi 1:.1:::11111 YVITASHCVNGKALPTDWRLSGVRLGEWDTNTNPDCEVDVRGMKDCAPPH * IRVTIDRILPHAQY..SELNYRNDIALLRLSRPVNWLQMQNLEPVCLPPQ : I:lIl IlIlllIl...l:: :: . : : l.:Il::I . . I ~DVPVERTIPH~DYIPASKNQVNDIALLRLAQQVEYTDF..VRPICLPLD RGRYANQLAGSAADVSGWGKTESSGSSKIKQKAMLHIQPQDQCQEAFYKD . . . :. I. II.IIIIll :.I.:) II :. . I:lI:.: VNLRSATFDGITMDVAGWGKTEQLSASNLKLKAAVEGSRMDECQNVYSSQ *
:
.:
TKITLADSQMCAGGEIGVDSCSGDSGGP. .I I.I.IIIIII. lllll.IIIIII .DILLEDTQMCAGGKEGVDSCRGDSGGPL
* WWTAAHCLDTPTTVSNLRIRAGSNKRTYGGVLVEVAAIKAHEAYNSNSK IIIIIIIII:...I I I: IIII. :III:I.III:I.II:II.... WVVTAAHCLQSVST SVLKARAGSSYWNSGGVWSVAAFKNHEGYNPKTM l
SP#3 LCU
INDIGWRLKTKLTFGSTIKAITMASATPAHGSAASISGWDKTSTDGPSS :lIl:l:lI...lI::IlllII.:..l.lI:I.II.:III:.II.:l. VNDIAVIRLSSSLTMSSTIKAIALTTAAPANGAAATVSGWGTTSSGGSIP
. *
SP#3 LCU
ATLLFVDTRIVGRSQCGSSTYGYGSFIKATMICAAATNKDACQGDSGGPL Il:.IIII . ..lI.lIIIIIIIl I I : I I :llll.ll:llllllll AQLRYVDLKIVGRTQCASSTYGYGSQIKPSMICAYTVGKDSCQGDSGGPL
Figure 2 Alignment of the deduced amino acid sequences of the 2 novel putative serine proteases gene fragments amplified from D. melanogaster (SP# 2, SP# 3) with the respective serine proteases corresponding to the highest ‘blast scores’. Identical residues are indicated by (I), and conserved residues by (:). EAST_DROM: D. melanogaster serine protease Easter precursor (23); LCUO76931:serine proteinase Lucilia cuprina (sbtrf in 21). Asterisks indicate the His-57, Asp-102 and Ser-195 residues characteristic of serine proteases.
by L.
Involvement of SPZ-related Gene in the Immune Response
boulardi, the expression of antibacterial
The stimulation of expression of SP2related gene in both S- and R-strain hosts following parasitic infection and bacterial exposure strongly suggests its involvement in the immune response, but raises the question of the apparent lack of specificity of this response. Although bacterial exposure elicited a significantly stronger induction of SP2 than did the parasite infection, we cannot specifically relate SP2 gene induction to the antibacterial response. Our previous studies
transcripts (cecropin and diptericin) is strongly induced in the S-strain, but not in the R-strain (20). Therefore, SP2 and antibacterial transcripts are not induced in a similar way. The induction of SP2 also does not seem to be directly correlated with phenoloxidase activity, since a strong increase in both monophenoloxidase and diphenoloxidase has been observed in hemolymph from the Rstrain 24 h after infection, but not in the hemolymph from the S-strain (25). Therefore, SP2 could participate in a reaction
showed that 24 h after infection
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S. strain
R. strain
c12c12
SP #l
SP #2
Figure 3. Leptopilina
Expression of SP# 1, SP# 2 and SP# 3 related gene transcripts in mRNA of control (C), boulardi-infected (1) and E. co/i-exposed (2) larvae from Drosophila S and R strains.
occurring systematically after stimulation of the internal defence system, independently of the type of stimulation and of the relative success or failure of the infection. Although the involvement of serine proteases in the insect immune response has only been linked with the activation of the PO system during melanotic reactions, there is a considerable potential for these enzymes also to participate immunologically in further pathways.
The extensive body of work carried out on mammalian immunogenetics reveals that serine proteases are involved in the regulation of diverse key reactions of the inflammation and acute phase responses taking place shortly after trauma or infections (26-28). Knowing that serine proteases are an evolutionary old and well-conserved enzyme family (5,29,30), and that they also represent a large gene family in insects (21), it seems likely that
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these enzymes may participate in a diversity of insects’ immune reactions. At present it is unclear as to exactly how SP2 is employed in the immune system of D. melanogaster, but its overexpression in immune challenged larvae suggests it may play a key role in this insect’s immune response.
Acknowledgements-We thank F. Shotkoski for his skilled assistance in the laboratory, S. Paskewitz for provision of the serine protease degenerate primers and critical reading of the manuscript, and T. Yoshino for comments on the manuscript. A. J. N. acknowledges support from the National Science Foundation (IBN 950 4796)
References 1. Boman, H. G. Antibacterial peptides: key components needed in immunity. Cell 65:205207; 1991. 2. Kimbrell, D. A. Insect antibacterial proteins: not just for insects and against bacteria. BioEssays 1312657-663; 1991. 3. Hultmark, D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 95:178-184; 1993. 4. Nappi, A. J.; Vass, E. Melanogenesis and the generation of cytotoxic molecules during insect cellular immune reactions. Pigment Cell Res. 6:117-126; 1993. 5. Neurath, H. Evolution of proteolytic enzymes. Science 224:35&357; 1984. 6. Dohke, K. Studies on prophenoloxidaseactivating enzyme from cuticle of the silkworm Bombyx mori. I. Activation reaction by the enzyme. Archs Biochem. Biophys. 157:203-209; 1973. 7. Sugumaran, M.; Saul, S. J.; Ramesh, N. Endogenous protease inhibitors prevent undesired activation of prophenoloxidase in insect hemolymph. Biochem. Biophys. Res. Comnmn. 13211241129; 1985. 8. Yoshida, H.; Ashida, M. Microbial activation of two serine enzymes and prophenoloxidase in the plasma fraction of hemolymph of the silkworm, Bombyx mori. Insect. Biochem. 163:539-545; 1986. 9. Ashida, M.; Kinoshita, K.; Brey, P. T. Studies on prophenoloxidase activation in the mosquito Aedes aegypti L. Eur. J. B&hem. 188:507-515; 1990. 10. Seybold, W. D.; Meltmr, P. S.; Mitchell, H. K. Phenol oxidase activation in Drosophila: a cascade of reactions. Biochem. Genet. 13:85108; 1975. 11. Rixki, T. M.; Rixki, R. M.; Bellotti, R. A. Genetics of a Drosophila phenoloxidase. Mol. Gen. Genet. 201:7-13; 1985. 12. Yonemura, M.; Kasatani, K.; Asada, N.; Oh&hi, E. Involvement of a serine protease in the activation of prophenoloxidase in Drosophila melanogaster. Zool. Sci. (Tokyo) 8:865-867; 1991. 13. Neurath, H. The versatility of proteolytic enzymes. J. Cell. Biochem. 32:35-49; 1986.
14. Baron, R. W.; Colwell, D. D. Mammalian immune responses to myasis. Parasitol. Today 7:353-355; 1991. 15. Elvin, C. M.; Whan, V.; Riddles, P. W. A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua). Mol. Gen. Genet. 240:132-139; 1993. 16. Casu, R. E.; Jarmey, J. M.; Elvin, C. M.; Eisemann, C. H. Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina. Insect Mol. Biol. 33:159-170; 1994. 17. Casu, R. E.; Pearson, R. D.; Jarmey, J. M.; Cadogan, L. C.; Riding, G. A.; Tellam, R. L. Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA. Insect Mol. Biol. 34:201-211; 1994. 18. Carton, Y.; Frey, F.; Nappi, A. Genetic determinism of the cellular immune reaction in Drosophila melanogaster. Heredity 69:393-399; 1992. 19. Carton, Y.; Nappi, A. The Drosophila immune reaction and the parasitoid capacity to evade it: genetic and coevolutionary aspects. Acta Ecol. 121389104; 1991. 20. Coustau, C.; Carton, Y.; Nappi, A. J.; Shotkoski, F. A. S.; ffrench-Constant, R. H. Differential induction of antibacterial transcripts in Drosophila susceptible and refractory to parasitism by Leptopilina boulardi. Insect Mol. Biol. 3:167-172; 1996. 21. Elvin, C. M.; Vuocolo, T.; Smith, W. J. M.; Eisemann, C. H.; Riddles, P. W. An estimate of the number of serine protease genes expressed in sheep blowtly larvae (Lucilia cuprina). Insect Mol. Biol. 32:105-115; 1994. 22. Brown, J. R.; Hartley, B. S. Location of disulphide bridges by diagonal paper electrophoresis. The disulphide bridges of bovine chymotrypsinogen. A. Biochem. J. 101:214228; 1966. 23. Chasan, R.; Anderson, K. V. The role of Easter, an apparent serine protease, in organizing the dorsal-ventral pattern of the Drosophila embryo. Cell 56z391-400; 1989.
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24. Yun, Y.; Davis, R. L. Levels of RNA from a family of putative serine protease genes are reduced in Drosophila nzelanogaster dunce mutants and are regulated by cyclic AMP. Mol. Cell. Biol. 92:692-700; 1989. 25. Nappi, A.; Carton, Y.; Frey, F. Parasiteinduced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster. Arch. Ins. Biochem. Biophys. 18:159-168; 1991. 26. Bamnann, H.; Gauldie, J. The acute phase response. Immunol. Today 152:74-80; 1994. 21. Vyse, T. J.; Bates, G. P.; Walport, M. J.; Morley, B. J. The organization of the human complement factor I gene (IF): a member of the
C. Coustau et al.
serine protease gene family. Genomics 2490-98; 1994. 28. Thia, K. Y. T.; Jenkins, N. A.; Gilbert, D. J.; Copeland, N. G.; Smyth, M. J. The natural killer cell serine protease gene Lmet 1 maps to mouse chromosome 10. Immunogenetics 41:4749; 1995. 29. Kraut, J. Serine proteases: structure and mechanism of catalysis. Ann. Rev. Biochem. 46:331-358; 1977. 30. Sakanari, J. A.; Staunton, C. E.; Eakin, A. E.; Craik, C. S.; McKerrow, J. H. Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. Proc. Natl. Acad. Sci. U.S.A. 8648634867; 1989.