- Email: [email protected]
The inducible antibacterial peptides of dipteran insects
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34 th F O R U M I N I M M U N O L O G Y
prophenoloxidase cascade components are present in granules of granulocytes, prophenoloxidase was clearly shown not to be present in granulocytes, but in oenocytoids and plasmatocytes. According to works of Sugumaran's group and ours, it is now evident that the prophenoloxidase cascade is present in the plasma fraction of insect haemolymph under normal physiological conditions (Ashida and Yamazaki, 1990, a review). The sites of synthesis of each cascade component and the mechanisms releasing these components, especially recognition proteins for microbial cell wall components, from haemocytes to plasma, remain to be studied. Another feature of the insect prophenoloxidase cascade is that it is present in haemolymph throughout the larval or nymphal and adult stages, almost the entire life span of the insect.
To take part in the initial defence response against non-self, defense molecules or systems must be present at the very mome:A, when the foreign body or substance enters the insect. In this respect, the insect prophenoloxidase cascade indeed takes part in the initial defense reaction against non-self. However, lectins znd immune proteins which have been shown to be synthesized after injury or invasion by microorganisms could not be molecules to initially detect foreignness in the insect, although a certain lectin is claimed to be involved in the recognition process at specific developmental stages of the insect. For a better comprehension of insect immunity, it seems necessary to understand how the various defence mechanisms function independently, as well as how they interact with one another.
References.
ASHIDA, M., IWAMA,R., IWAHANA,H. & YOSHIDA,H. (1982), Control and function of the prophenoloxidase activating system, in Proc. 3rd Int. Collog. Invertebrate Pathology (pp. 81-86). University of Sussex, Brightop ASHIDA,M. & YAMAZAK!,H.I. 0990), Biochemistry of the phenoloxidase system'in insects: with special reference to its activation, in "Molting and metamorphosis" (Ohaishi, E. and Ishizaki, H.) (pp. 237-263). Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin. LACKIE,A.M. (1988), Hemocyte behaviour. Advanc. Insect Physio.'., 21, 85-178. SODERH~LL,K. (1982), Prophenoloxidase activating system and melanization. -- A recognition mechanism of arthropods? A review. Develop. Comp. Immunol., 0, 601..611.
THE INDUCIBLE ANTIBACTERIAL PEPTIDES OF DIPTERAN INSECTS J.A. Hoffmann and D. Hoffmann
Laboratoire de Biologie Gdndrale, Univer~itd Louis P a s t e u r - URA CNRS 672, 12, rue de l'Universit~, 67000 Strasbourg (France)
The synthesis of antibaeterie! substances by injured insects has attracted considerable interest after the pioneer-
ing studies of Hans Boman and associates in Stockholm. During the early eighties, these authors have indeed iso-
INSECT IM3/[UNITY
lated from stimulated ("immune") pupae of the moth Hyalophora cecropia two families of novel antibacterial peptides, the cecropins (six isoforms of = 4 kDa; Hultmark et al., !980, 1982; Steiner et al., 1981) and the attacins (six isoforms of basic or acidic peptides of 20 to 23 kDa, Hultmark et al., 1983; Kockum et al., 1984). Together with in~ sect lysozyme (Powning and Davidson~ 1976; Engstr6m et al., 1985), the cecropins and attacins account for the major part of the immune humoral response of H. cecropia. The present contribution is devoted to the inducible antibacterial peptides of dipteran insects. Three species have been the object of intense and continuing interest: Sarcophaga peregrina (Natori and associates, Tokyo), Phormia terranovae (this laboratory, Strasbourg) and Drosophila melanogaster (Hultmark and associates, Stockholm; this laboratory, Strasbourg).
91 i
sized as a 12-kDa prediptericin (fig. 2), as deduced from cDNA clones isolated from libraries prepared from mRNA of immune larvae. The precursor comprises a signal sequence of 18 residues plus the 82 amino acids of mature dipl tericin. The presence of a glycine codon at the 3' end of the coding sequence in diptericin eDNA clones indicates that Phormia diptericin is amidated (Reichhart et al., 1989). Southern hybridization experiments using gen~mic DNA of single larvae suggest that there are multiple copies of diptericin genes in the genome of Pht~rmia (unpublished). Studies with semi-purified diptericin indicate that they exert their activity at the level of the cytoplasmic membrane ir~ Gram-negative bacteria (Keppi et ai., 1989). / Defensins.
Structures of the inducible antibacterial peptides Phormia terranovae.
Injection of heat-killed bacteria into third instar larvae results in the rapid (6..10 h) appearance in the haemolymph of a strong antibacterial activity (Keppi et al., 1986). Several molecules responsible for this activity have now been fully characterized. Diptericins.
These are the predominant antiGram-negative peptides of immune Phormia larvae. Three diptericins have been isolated and the amino acid sequence has been completely established for the major one, diptericin A. The seuences show marked homology inicating that the three peptides belong to a common family (Dimarcq et aL, 1988; fig. 1).
q
Diptericin A of Phormia is an 82-residue basic peptide which is synthe-
Immune haemolymph of Phormia contains two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues differing from one another by only a single amino acid (fig. 3). These peptides show significant homology with a family of small cationic peptides which are highly abundant in the granules of mammalian phagocytes, the defensins (review in Ganz et al., 1990), and were consequently named insect defensins (Lambert et al., 1989). Direct measurement of the molecular mass of defensin A and comparison with the mass given by the amino acid sequence indicates that defensin A is not posttranslationally modified and that the six cysteines are involved in three disulphide bridges (Lambert et al., 1989). The position of these blidges has been attributed as: C3-C30, C16-C36, C20-C38 (Lepage et aL, 1990). Phormia defensin is produced as a 10-kDa prepropeptide yielding mature defensin (40 residues) after cleavage of a putative signal peptide (23 residues) and a prosequence (34 residues) (fig. 2) (Dimarcq et al., 1990).
912
Dip A
34 th F O R U M
1 ~E
IN IMMUNOLOGY
I0 K P K L I L P~T~A
P~N
L P~L
20 V
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D E K P K L V L P(S)X A P P N L P Q L V
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30
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40 KVWT
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61
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8O
7O
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:bF F
70 F P N F sT Z TIY R i'°
FI6. 1. -- Amino acid sequences of diptericins from different species of Diptera. D.m. = D. melanogaster. Conserved residues are boxed. Diptericin A was totally (diptericins B and C only partially) purified from immune haemolymph of P. terranovae; diptericins D and D.m. were deduced from cDNA nucleotidic sequences.
iNSECT IMMUNITY
[:o:................
....DIPT(82)
9!
I .AU
Dipleridn P. L
Dipiedcin D. m.
:.: S(23) =.::'.I~ .:. ?! PRO(31) ~,~I ;~'.'..-..-..-.... ~%~,~,~,=,, ,,~.~,~.-:~.-~
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....
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FIG. 2.
- -
Sarcotoxin I S.p.
SARC,12eg)
¢
,, ¢
Processing of immune peptides, deduced from cDNA of different species of Diptera.
P.t. = P. terranovae; D.m. = D. melanogaster; S.p. = S. peregrina. The ::umbers in brackets indicate the length (in amino acids) of the corresponding peptidic fragment: $ = signal peptide; PRO = propeptide; DIPT = diptericin; DEF = defensin; SARC l = sarcotoxin I ; SARC II: sarcotoxin II.
1
10
20
Def A
A T C D L L S G T G I N H S A C A A H C
Def B
A T C D L L S G T G I N H S A C A A H C
Sapecln
A T C D L L ~ G T G I N H S A C A A H C 21
30
40
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L L R G N R G G Y C N G K G V C V C R N
Def B
LLRGNRGG¥CNRKGVCVCRN
Snpecln
L L R G N R G G ¥ C N G K A V C V ~ R N
FIG. 3. -- Amino acid sequences of the insect defensins A and B (from P. terranovae) and of the related Sapecin (from a S. peregrina embryonic cell line culture). Bold letters correspond to the variable residues in the three insect defensins.
Cecropins. I m m u n e haemolymph of Phormia contains cecropins which we have purified to homogeneity. The sequence of the first 10 N-terminal residues (fig. 4) shows perfect identity with the cot-
responding residues of sarcotoxias I of S. peregrina (see below). S, peregrina. Several antibacteri~ peptides of imm u n e Sarcophaga have been character-
914
34 th F O R U M I N I M M U N O L O G Y Cecroplns 1
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FIG. 4. -- Amino acM sequences of members of the cecropin family in differe.~t species o f insects.
S.p. = S. peregrina; D.m. =D. melanogaster; H.c. = H. cecropia (Lepidoptera). PhV (from P. terranovae) has only been partially characterized.
ized over the last years by Natori and associates. These authors refer to the active substances as sarcotoxins, adding the suffix I, II, III, in the chronological order of the structural characterization. Sarcotoxins I (members o f the cecropin family).
A first group of three potent antibacterial peptides (active on Gramnegative and Gram-positi:~e cells) was isolated from immune larvae of Sarcophaga and named sarcotoxins IA, IB, IC (fig. 4) (Okada and Natori, 1985a). They show an almost identical primary structure of 39 residues differing only by two or three replacements. The primary structures of sarcotoxins I are very close to those of the cecropins of
H. cecropia initially characterized by Boman and associates (Hultmark et al., 1980; Steiner et al., 1981). Sarcotoxin IA is synthesized as a 63-residue prepeptide (fig. 2). The C-terminal amino acid residue is amidated (Matsumoto et aL, 1986) as in H. cecropia (van Hofsten et al., 1985). The primary target of sarcotoxins I is the cytoplasmic membrane. On treatment wkh se~cotoxin IA, the membrane potential of E. coli disappears almost instantaneously, resulting in loss of active transport of amino acids such as proline and of ATP generation (Okada and Natori, 1985b). Sarcotoxins II.
Sarcotoxins II represent a group of three structurally related proteins, IIA,
INSECT IMMUNITY IIB and IIC, each with a MW of 24 kDa (Ando et al., 1987). Molecular cloning of eDNA indicates that sarcotoxin IIA consists of 270 residues (Ando and Natori, 1988) (fig. 2). The overall deduced amino acid sequence is relatively different from that of attacins (Kockum et al., 1984). However, significant homology is observed between these proteins in a certain region near the carboxyl terminus. Hybridization of genomic DNA with a sarcotoxin IIA probe indicates that sarcotoxins II are a multigene family. The presence of a glycine sequence at the C-terminus of sarcoto×Jn IIA eDNA clones suggests that sarcotoxin II is Camidated (Ando and Natori, 1988).
Sarcotoxin IlL A glycine-rich antibacterial protein with a molecular mass of 7 kDa, termed sarcotoxin III, was purified to homogeneity fi'om immune haemolymph of third instar larvae of Sarcophaga. It is clearly different from sarcotox~as I and II in amino-acid composition and molecular mass (Baba et al., 1987). The sequence remains to be established.
Sapecin (insect defensin). From an embryonic cell line of
S. peregrina, an antibacterial protein was isolated ,rod characterized (Matsuyama and Natori, 1988a). It consists of 40 residues with near sequence identity to insect defensin A from immune P. terranovae (replacement of a Gly by an Ala in position 34) and has three disulphide bridges in positions identical to those of the Phormia counterpart (Kuzuhara et al., 1990). A eDNA clone of Sarcophaga sapecin was isolated and found to encode a precursor of 94 residues with sapecin (40 residues) constituting its carboxyl-half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the fat body and some haemocytes of third instar larvae of Sarcophaga in response to body injury (Matsuyama and Natori,
915
1988b). Interestingly, it is also expressed during normal development in late embryogenesis and in early pupae (see the contribution by S. Natori in this volume).
Drosophila melanogaster. The main characteristics of the immune response of Drosophila have been investigated by several authors (Eakula, 1970; Boraan et al., 1972; Robertson and Postlethwait, 1986; Flyg et aL, 1987), whose results pointed to many similarities with other holometabolous insects. Essentially because of the small size of Drosophila larvae and adults, biochemical characterization of immune peptides has not been successfully performed so far. The existence of Drosophila homologues of cecropins (sarcotoxins I) and diptericins has recently been ascertained with the use of nucleotide probes encoding these molecules respectively in Sarcophaga and Phormia.
Cecropins. Hultmark and associates, using a cDNA clone corresponding to the major cecrtJpin of Sarcophaga (sarcotoxin IA), have characterized in the genome of Drosophila three functional cecropin genes and two pseudogenes, all ar~ ranged as a compact cluster within less than 4 kb of DNA in the genome. Two of the genes - - named A1 and A2 encode a product that is identical to sarcotoxin IA from Sarcophaga (see above), while the cecropin encoded by the third gene. B, differs in five positions (Kylsten et al., 1990) (fig. 4). All three forms are C-ter. -inally amidated. Cecropin transcripts appear rapidly after bacteria ha~ e been injected into the haemocoel of Drosophila and cecropin is identified in the blood of immunized flies at a significant concentration of 25 to 50 I~M. A small peak of constitutive expression in early pupae appears to be caused by bacteria in the food (Samakovlis et al., 1990).
34 th F O R U M I N I M 3 , 1 U N O L O G Y
916
Diptericins. From a cDNA library of immune Drosophila, Wicker et al. (1990) have isolated, with an oligonucleotide probe corresponding to part of Phormia diptericin, a clone encoding a peptide structurally related to this antibacterial molecule. The deduced primary sequence of the Drosophila diptcricin shows an overall homology of approximately 60 % with that of the Phormia peptide with several regions (of 4 residues and more) of full homology (fig. 1). In particular, a pentagiycine stretch characteristic of the Phormia diptericins, is present in an equivalent relative position in Drosophila. Transcripts for the Drosophila diptericin are detected 2 h after injection of bacteria. The hybridization signal increases to its maximum at 4 h after which it becomes fainter. Interestingly, unchallenged early pupae and adults appear to contain low but significant amounts of diFtcricin transcripts which are however not detected in embryos or larvae (Wicker et al., 1990). In comparison to cecropin transcripts, the diptericin transcripts are found to be very abundant in immune ;ncpptc /l¢'vlctpn ot nl
IQOfi" W ; ~ l ~ r
ot
al., 1990). L ysozyme. Flyg and associates (1987) have detected lysozyme activity in Drosophila larvae and recently, Hultmark and coworkers have cloned the lysozyme locus in this species (Kylsten et al., personal communication). Functional lysozyme has not been detected in Phormia (unpublished results from this laboratory).
Expression of inducible antibacterial peptides The studies on Sarcophaga, Phormia and Drosophila conclusively point to the fat body as a major site of biosynthesis for the inducible antibacterial peptides. The role of the haemocytes is less well established, which is indeed not
surprising as they represent several cell lineages which are apparently not fully superposable in Drosophila and Phormia. We have recently compared in Phormia the expression of the predominant anti Gram-negative peptides in this species, i.e. the diptericins, and that of the major anti-Gram-positive molecules, the defensins (Dimarcq et al., 1990). The results show that all cells of the fat body express the genes coding for both peptides in a concomitant manner. In addition to the fat body cells, one single blood cell type, the thrombocytoids, also express concomitantly both types of genes. Thrombocytoids are remarkable blood cells in many respects, which undergo partial fragmentation during defence reactions, agglutinate around bacteria and seal off wounds of the integument. Interestingly, plasmatocytes which account for 80 % of the 10,000 to 12,000 cells per mm3 of blood of wandering larvae and are strongly phagocytic, do not express the genes ~or diptericins and defensins. Sarcophaga also contains thrombocytoids and, although this has not been specifically investigated, we assume that the undefined haemoc~es shown by ~.he Tokyo group to express defensin genes (sapecin) in Sarcophaga larvae correspond to this blood cell category (Matsuyama and Natori, 1988b). In contrast, Drosophila apparently lacks thrombocytoids. We did not observe expression of the diptericin gene in any Drosophila blood cell (unpublished), while Hultmark and associates monitored expression of the cecropin genes in some 10 % of the circulating haemocytes. These cells probably correspond to lamellocytes (Samakovlis et al., 1990). Studies based on transcriptional profiles have shown in Phormia that the concomitant expression of the genes encoding the anti-Gram-positive defensins and the anti-Gram-negative diptericins is independent from the nature of the stimulus: live or heat-killed Grampositive or Gram-negative bacteria, lipopolysaccharides, distilled water, induce the ev,~ression of both types of genes. Even a simple injury of axenical-
INSECT
ly raised larvae performed under strict sterile conditions is sufficient to induce the expression of diptericin and defensin genes in Phormia, suggesting that the disruption of the integument releases a signal or initiates a cascade of biochemical events, inducing the transcription of the genes encoding the antibacteria! peptides. When the response to a second stimulus was investigated in larvae which had been challenged by a first injection, it was found that the two responses were equivalent. This result does not argue in favour of a mechanism allowing the insect to keep a " m e m o r y " of the first injection (Dimarcq et al., 1990). In eonelusion, the humoral immune response of the three dipteran insects is primarily based on the rapid and transient transcription of a battery of genes encoding mostly small cationic antibacterial peptides. This response is a nonadaptative process which lacks both specificity and memory. The recent cloning of the genes encoding cecropins, diptericins and lysozyme of Drosophila shouid now enable *~-'~dissection of ,the control of the tran-
IMMUNITY
917
scription of these genes in this favourable biological model. In this context, the use of various cell lines should prove particularly rewarding. The signalling mechanism informing the adipocytes and blood cells of the injury may turn out to be more difficult to unravel, although some experimental models are now at hand. Of great interest are also the observations that some of the antibacterial peptides are synthesized during pupal development. These results point to a possible link between the endocrine system (control of metamorphosis) and the immune system in holometabolous insects (see contribution of Natori, this volume). As the antibacterial peptides of Diptera have now been either chemically synthesized (see Samakovlis et al., 1990 for Drosophila cecropins) or their cDNA expressed in heterologous systems (see Reichhart ar,d Achstetter, this volume, for Phormia defensin), the analysis of their mode of action and the structure-function relationship will be amenable to thorough investigations. Some of these molecules may eventually prove of interest in various fields of biotechnology (see Casteels, this Forum).
References.
ANDO, K. et ai. (1987), Purification of Sarcotoxin lI, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae. Biochemistry, 26, 226-230. ANDO,K. & NAa'ORI,S. (1988), Molecular cloning, sequencing and characterization of cDNA for Sarcotoxin IIA, an inducible antibacterial protein of Sercophagaperegrina (flesh fly). Biochemistry, 27, 1715-1721. BABA,K. et al. (1987), Purification of Sarcotoxin III, a new antibacterial protein of Sarcophaga peregrina. J. Biochem., 102, 69-74. BAKtJLA, M. (1970), Antibacterial compounds in the cell-free haemolymph of Drosophila melanogaster. J. Insect PhysioL, 16, 185-197. BOMAN, H.G. et al. (1972), Inducible antibacterial defence system in Drosophila. Nature (Lond.), 237, 232-234. DIMARCQ,J.L. et al. (1988), Insect Immunity. Purification and characterization of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino acid sequence of the predominant member, diptericin A. Europ. J. Biochem., 171, 17-22. D1MARCQ,J.L. et al. (1990), Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae. EMBO J., 9, 2507-2515. Er~csTgOM,A. et al. (1985), Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia. EMBO J., 4, 2119-2122. FLVC,C. et al. (1987), Insect Immunity. Inducible antibacterial activity in Drosophila. Insect Biochem., 17, 151-160.
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34 th F O R U M I N I M M U N O L O G Y
GANZ, T. et al. (1990), Defensins. Europ. J. Haematol., 44, 1-8. HULTMARK,D. et al. (1980), Insect Immunity: purification and properties of thr~.e inducible bactericidal proteins from haemolymph of immunized pupae of Hyalophora cecropia. Europ. J. Biochem., 106, 7-16. HULTMARK,D. et al. (1982), Insect Immunity. Isolation and structure of cecropin D and four minor antibacterial components from cecropia pupae. Europ. J. Biochem., 127, 207-217~ HULTMARK,D. et al. (1983), Insect Immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J., 2, 571-576. KEpPI, E. et al. (1986), Induced antibacterial proteins in the haemolymph of Phormia terranovae (Diptera). Insect Biochem., 16, 395-402. KEPPh E. et al. (1989), Mode of action of diptericin A, a bactericidal peptide induced in the hemolymph of Phormia terranovae larvae. Arch. Insect Biochem. Physiol., 10, 229-239. KOCKUM,K. et al. (1984), Insect Immunity: isolation and ~equence of two cDNA clones corresponding to acidic and basic attacins of Hyalophora cecropia. EMBO J., 3, 207!-2075. KUZUHARA,T. et al. (1990), Determination of the disulfide array in Sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly). J. Biochem., 107, 514-518. KYLSTEN,P. et al. (1990), The cecropin locus in Drosophila, a compact gene cluster involved in the respt~nse to infection. EMBO J., 9, 217-224. LAMaEP.% J. et al. (1989), Insect Immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proc. nat. Acad. Sci. (Wash.), 86, 262-266. LEPAGE,P. et al. (1990), Determination of disulfide bridges in natural and recombinant insect defensin A. Europ. J. Biochem. (submitted). MATSUMOTO,hi. et al. (1986), Molecular cloning of cDNA and assignement of the C-termipal of Sarcotoxin IA, a potent antibacterial protein from Sarcophaga peregrina. Biochem. J., 239, 717-722. MATSUYAMA,K. & NATORI,S. (1988a), Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. J. biol. Chem., 263, 17112-17116. MATSUYAMA,K. & NATORI,S. 0988b), Molecular cloning of cDNA for Sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina. J. biol. Chem., 263, 17117-17121. OKADA,M. & NATORI,S. (1985a), Primary structure of Sarcotoxin I from Sarcophagaper~riha. J. biol. chem., 260, 7174~7177. OKADA,M. ~ NATORI,S. (1985b), Mode of action of a bactericidal protein induced in the haemolymph of Sarcophaga peregrina (flesh-tly) larvae. Biochem. J., 222, 119-124. POWN~NG,R.F. & DAWOSON,W.J. (1976), Studies on insect bacteriolytic enzymes. -- II. Some physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella. Comp. Biochem. Physiol., 55B, 221-228. REICHHART,J.M. et al. (1989), In~ect Immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera). Transcriptional profiles during immunization. Europ. J. Biochem., 182, 423-427. ROBERTSON,M. & POSTI.ETHWAIT,J.H. (1986), The humoral antibacterial response of Drosophila adults. Dev. Comp. Jmmunol., 10, 167-179. SAMAKOVLIS~C. et al. (1990), The immune response in Drosophila: pattern of cecropin expression and biological activity. EMBO J., 9, 2969-2976. STS~NER,H. et al. (1981), Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature (Lond.), 292, 246-248. VAN HOFSTEN,P. et al. (1985), Molecular cloning, cDNA sequencing and chemical synthesis of cecropin B from Hyalophora cecropia. Proc. nat. Acad. Sci. (Wash.), 82, 2240-2243. WICrER, C. et al. (1990), Insect immunity. Characterization of a Drosophila cDNA encoding a novel member of the diptericin family of immune peptides. J. biol. Chem. (in press).
34 th F O R U M I N I M M U N O L O G Y
prophenoloxidase cascade components are present in granules of granulocytes, prophenoloxidase was clearly shown not to be present in granulocytes, but in oenocytoids and plasmatocytes. According to works of Sugumaran's group and ours, it is now evident that the prophenoloxidase cascade is present in the plasma fraction of insect haemolymph under normal physiological conditions (Ashida and Yamazaki, 1990, a review). The sites of synthesis of each cascade component and the mechanisms releasing these components, especially recognition proteins for microbial cell wall components, from haemocytes to plasma, remain to be studied. Another feature of the insect prophenoloxidase cascade is that it is present in haemolymph throughout the larval or nymphal and adult stages, almost the entire life span of the insect.
To take part in the initial defence response against non-self, defense molecules or systems must be present at the very mome:A, when the foreign body or substance enters the insect. In this respect, the insect prophenoloxidase cascade indeed takes part in the initial defense reaction against non-self. However, lectins znd immune proteins which have been shown to be synthesized after injury or invasion by microorganisms could not be molecules to initially detect foreignness in the insect, although a certain lectin is claimed to be involved in the recognition process at specific developmental stages of the insect. For a better comprehension of insect immunity, it seems necessary to understand how the various defence mechanisms function independently, as well as how they interact with one another.
References.
ASHIDA, M., IWAMA,R., IWAHANA,H. & YOSHIDA,H. (1982), Control and function of the prophenoloxidase activating system, in Proc. 3rd Int. Collog. Invertebrate Pathology (pp. 81-86). University of Sussex, Brightop ASHIDA,M. & YAMAZAK!,H.I. 0990), Biochemistry of the phenoloxidase system'in insects: with special reference to its activation, in "Molting and metamorphosis" (Ohaishi, E. and Ishizaki, H.) (pp. 237-263). Japan Sci. Soc. Press, Tokyo/Springer-Verlag, Berlin. LACKIE,A.M. (1988), Hemocyte behaviour. Advanc. Insect Physio.'., 21, 85-178. SODERH~LL,K. (1982), Prophenoloxidase activating system and melanization. -- A recognition mechanism of arthropods? A review. Develop. Comp. Immunol., 0, 601..611.
THE INDUCIBLE ANTIBACTERIAL PEPTIDES OF DIPTERAN INSECTS J.A. Hoffmann and D. Hoffmann
Laboratoire de Biologie Gdndrale, Univer~itd Louis P a s t e u r - URA CNRS 672, 12, rue de l'Universit~, 67000 Strasbourg (France)
The synthesis of antibaeterie! substances by injured insects has attracted considerable interest after the pioneer-
ing studies of Hans Boman and associates in Stockholm. During the early eighties, these authors have indeed iso-
INSECT IM3/[UNITY
lated from stimulated ("immune") pupae of the moth Hyalophora cecropia two families of novel antibacterial peptides, the cecropins (six isoforms of = 4 kDa; Hultmark et al., !980, 1982; Steiner et al., 1981) and the attacins (six isoforms of basic or acidic peptides of 20 to 23 kDa, Hultmark et al., 1983; Kockum et al., 1984). Together with in~ sect lysozyme (Powning and Davidson~ 1976; Engstr6m et al., 1985), the cecropins and attacins account for the major part of the immune humoral response of H. cecropia. The present contribution is devoted to the inducible antibacterial peptides of dipteran insects. Three species have been the object of intense and continuing interest: Sarcophaga peregrina (Natori and associates, Tokyo), Phormia terranovae (this laboratory, Strasbourg) and Drosophila melanogaster (Hultmark and associates, Stockholm; this laboratory, Strasbourg).
91 i
sized as a 12-kDa prediptericin (fig. 2), as deduced from cDNA clones isolated from libraries prepared from mRNA of immune larvae. The precursor comprises a signal sequence of 18 residues plus the 82 amino acids of mature dipl tericin. The presence of a glycine codon at the 3' end of the coding sequence in diptericin eDNA clones indicates that Phormia diptericin is amidated (Reichhart et al., 1989). Southern hybridization experiments using gen~mic DNA of single larvae suggest that there are multiple copies of diptericin genes in the genome of Pht~rmia (unpublished). Studies with semi-purified diptericin indicate that they exert their activity at the level of the cytoplasmic membrane ir~ Gram-negative bacteria (Keppi et ai., 1989). / Defensins.
Structures of the inducible antibacterial peptides Phormia terranovae.
Injection of heat-killed bacteria into third instar larvae results in the rapid (6..10 h) appearance in the haemolymph of a strong antibacterial activity (Keppi et al., 1986). Several molecules responsible for this activity have now been fully characterized. Diptericins.
These are the predominant antiGram-negative peptides of immune Phormia larvae. Three diptericins have been isolated and the amino acid sequence has been completely established for the major one, diptericin A. The seuences show marked homology inicating that the three peptides belong to a common family (Dimarcq et aL, 1988; fig. 1).
q
Diptericin A of Phormia is an 82-residue basic peptide which is synthe-
Immune haemolymph of Phormia contains two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues differing from one another by only a single amino acid (fig. 3). These peptides show significant homology with a family of small cationic peptides which are highly abundant in the granules of mammalian phagocytes, the defensins (review in Ganz et al., 1990), and were consequently named insect defensins (Lambert et al., 1989). Direct measurement of the molecular mass of defensin A and comparison with the mass given by the amino acid sequence indicates that defensin A is not posttranslationally modified and that the six cysteines are involved in three disulphide bridges (Lambert et al., 1989). The position of these blidges has been attributed as: C3-C30, C16-C36, C20-C38 (Lepage et aL, 1990). Phormia defensin is produced as a 10-kDa prepropeptide yielding mature defensin (40 residues) after cleavage of a putative signal peptide (23 residues) and a prosequence (34 residues) (fig. 2) (Dimarcq et al., 1990).
912
Dip A
34 th F O R U M
1 ~E
IN IMMUNOLOGY
I0 K P K L I L P~T~A
P~N
L P~L
20 V
Dip B
D E K P K L V L P(S)X A P P N L P Q L V
DIP C
DEKPKL
DIP D
[~:
I XP
K P K L I L~I~A
I~N
MTMKPT
DIP D.m.
Q
21 DIP A
XXAPXNLXQLV L P~L LNL
30
39
tG e e G G~N R KID G F G[V S ~ D
DIP B
GGGGGNNKXGXXVS
DIP C
G GGG
GNN
V
A ~
INXAQ
KK
XXGVXV
XAAQ
DIP D G G G G Q S G D G
DIP D.m.
F A
Q G[H Q
50
DIP A
40 KVWT
SDN
G GIH
DIP D
KVWT
S DN
G
DIP D.m.
40 KVWT
S D N G RH
61
60
S I GVT
P GY
50 E I.._GGL N C- G Y
GP
YGN
S
DIP D
GP
YGN
S R
D Y R I ~G~A
6(3 G GP
YGN
S
SwKv
DIP D.m.
LQ H
8O
7O
DIP A
RID
QH
YR
I G A ~ YY~ S!Y
:bF F
70 F P N F sT Z TIY R i'°
FI6. 1. -- Amino acid sequences of diptericins from different species of Diptera. D.m. = D. melanogaster. Conserved residues are boxed. Diptericin A was totally (diptericins B and C only partially) purified from immune haemolymph of P. terranovae; diptericins D and D.m. were deduced from cDNA nucleotidic sequences.
iNSECT IMMUNITY
[:o:................
....DIPT(82)
9!
I .AU
Dipleridn P. L
Dipiedcin D. m.
:.: S(23) =.::'.I~ .:. ?! PRO(31) ~,~I ;~'.'..-..-..-.... ~%~,~,~,=,, ,,~.~,~.-:~.-~
DEF(40}
5e~'ensin P. t
....
SARC I (39) __ [.AM
:::::"s
:i::::::
• " : . • . ~ . • . • ._.-'.:'0"
FIG. 2.
- -
Sarcotoxin I S.p.
SARC,12eg)
¢
,, ¢
Processing of immune peptides, deduced from cDNA of different species of Diptera.
P.t. = P. terranovae; D.m. = D. melanogaster; S.p. = S. peregrina. The ::umbers in brackets indicate the length (in amino acids) of the corresponding peptidic fragment: $ = signal peptide; PRO = propeptide; DIPT = diptericin; DEF = defensin; SARC l = sarcotoxin I ; SARC II: sarcotoxin II.
1
10
20
Def A
A T C D L L S G T G I N H S A C A A H C
Def B
A T C D L L S G T G I N H S A C A A H C
Sapecln
A T C D L L ~ G T G I N H S A C A A H C 21
30
40
Def A
L L R G N R G G Y C N G K G V C V C R N
Def B
LLRGNRGG¥CNRKGVCVCRN
Snpecln
L L R G N R G G ¥ C N G K A V C V ~ R N
FIG. 3. -- Amino acid sequences of the insect defensins A and B (from P. terranovae) and of the related Sapecin (from a S. peregrina embryonic cell line culture). Bold letters correspond to the variable residues in the three insect defensins.
Cecropins. I m m u n e haemolymph of Phormia contains cecropins which we have purified to homogeneity. The sequence of the first 10 N-terminal residues (fig. 4) shows perfect identity with the cot-
responding residues of sarcotoxias I of S. peregrina (see below). S, peregrina. Several antibacteri~ peptides of imm u n e Sarcophaga have been character-
914
34 th F O R U M I N I M M U N O L O G Y Cecroplns 1
10
20
Wh V
G~KKTGKKI
S.p.Ia
GWLKK~GIKIERVGOH~RDA
$.p. :b
GWLKMICFKIERVG~HTRD~
S.p..'c
~W~RKIGEK!ERVG~HTRDA
D.m,&1
GWDKKZGKKZERVGQHTROA
D.~,A2
GWLKKIGKKIERVGQHT~DA
D.m B
G~LRKLGEKI£RIGQ~TRDA
H.C.A
KWKLFKKIEKVGQNIRDGII
H.~.B
KWKVFKKIEKMGRNTRNGIV
H.~,D
WNPFKELEKVGQRVRDAVV
21
~0
" 39
S.p.la
TIQGLGIAQQAANVAATAR
S.p.lb
TIQVIGVAQQAANVAAT~R
S.p.Ic
TIQVLGIAQQAANV~ATAR
D.m.:l
TIQGLGIAQQAANVAATAR
D,m.~
TIQGL~IAQQA&NVAATAR
D.m.B
SIQVLGIAQQAAHVAATAR
H.C.~
K~GPAVAVVGGATQIAK
H.¢.B
KAGPAIAVLGE&KAILS
H.c.D
SAGFAVATVAQ&T&LAK
FIG. 4. -- Amino acM sequences of members of the cecropin family in differe.~t species o f insects.
S.p. = S. peregrina; D.m. =D. melanogaster; H.c. = H. cecropia (Lepidoptera). PhV (from P. terranovae) has only been partially characterized.
ized over the last years by Natori and associates. These authors refer to the active substances as sarcotoxins, adding the suffix I, II, III, in the chronological order of the structural characterization. Sarcotoxins I (members o f the cecropin family).
A first group of three potent antibacterial peptides (active on Gramnegative and Gram-positi:~e cells) was isolated from immune larvae of Sarcophaga and named sarcotoxins IA, IB, IC (fig. 4) (Okada and Natori, 1985a). They show an almost identical primary structure of 39 residues differing only by two or three replacements. The primary structures of sarcotoxins I are very close to those of the cecropins of
H. cecropia initially characterized by Boman and associates (Hultmark et al., 1980; Steiner et al., 1981). Sarcotoxin IA is synthesized as a 63-residue prepeptide (fig. 2). The C-terminal amino acid residue is amidated (Matsumoto et aL, 1986) as in H. cecropia (van Hofsten et al., 1985). The primary target of sarcotoxins I is the cytoplasmic membrane. On treatment wkh se~cotoxin IA, the membrane potential of E. coli disappears almost instantaneously, resulting in loss of active transport of amino acids such as proline and of ATP generation (Okada and Natori, 1985b). Sarcotoxins II.
Sarcotoxins II represent a group of three structurally related proteins, IIA,
INSECT IMMUNITY IIB and IIC, each with a MW of 24 kDa (Ando et al., 1987). Molecular cloning of eDNA indicates that sarcotoxin IIA consists of 270 residues (Ando and Natori, 1988) (fig. 2). The overall deduced amino acid sequence is relatively different from that of attacins (Kockum et al., 1984). However, significant homology is observed between these proteins in a certain region near the carboxyl terminus. Hybridization of genomic DNA with a sarcotoxin IIA probe indicates that sarcotoxins II are a multigene family. The presence of a glycine sequence at the C-terminus of sarcoto×Jn IIA eDNA clones suggests that sarcotoxin II is Camidated (Ando and Natori, 1988).
Sarcotoxin IlL A glycine-rich antibacterial protein with a molecular mass of 7 kDa, termed sarcotoxin III, was purified to homogeneity fi'om immune haemolymph of third instar larvae of Sarcophaga. It is clearly different from sarcotox~as I and II in amino-acid composition and molecular mass (Baba et al., 1987). The sequence remains to be established.
Sapecin (insect defensin). From an embryonic cell line of
S. peregrina, an antibacterial protein was isolated ,rod characterized (Matsuyama and Natori, 1988a). It consists of 40 residues with near sequence identity to insect defensin A from immune P. terranovae (replacement of a Gly by an Ala in position 34) and has three disulphide bridges in positions identical to those of the Phormia counterpart (Kuzuhara et al., 1990). A eDNA clone of Sarcophaga sapecin was isolated and found to encode a precursor of 94 residues with sapecin (40 residues) constituting its carboxyl-half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the fat body and some haemocytes of third instar larvae of Sarcophaga in response to body injury (Matsuyama and Natori,
915
1988b). Interestingly, it is also expressed during normal development in late embryogenesis and in early pupae (see the contribution by S. Natori in this volume).
Drosophila melanogaster. The main characteristics of the immune response of Drosophila have been investigated by several authors (Eakula, 1970; Boraan et al., 1972; Robertson and Postlethwait, 1986; Flyg et aL, 1987), whose results pointed to many similarities with other holometabolous insects. Essentially because of the small size of Drosophila larvae and adults, biochemical characterization of immune peptides has not been successfully performed so far. The existence of Drosophila homologues of cecropins (sarcotoxins I) and diptericins has recently been ascertained with the use of nucleotide probes encoding these molecules respectively in Sarcophaga and Phormia.
Cecropins. Hultmark and associates, using a cDNA clone corresponding to the major cecrtJpin of Sarcophaga (sarcotoxin IA), have characterized in the genome of Drosophila three functional cecropin genes and two pseudogenes, all ar~ ranged as a compact cluster within less than 4 kb of DNA in the genome. Two of the genes - - named A1 and A2 encode a product that is identical to sarcotoxin IA from Sarcophaga (see above), while the cecropin encoded by the third gene. B, differs in five positions (Kylsten et al., 1990) (fig. 4). All three forms are C-ter. -inally amidated. Cecropin transcripts appear rapidly after bacteria ha~ e been injected into the haemocoel of Drosophila and cecropin is identified in the blood of immunized flies at a significant concentration of 25 to 50 I~M. A small peak of constitutive expression in early pupae appears to be caused by bacteria in the food (Samakovlis et al., 1990).
34 th F O R U M I N I M 3 , 1 U N O L O G Y
916
Diptericins. From a cDNA library of immune Drosophila, Wicker et al. (1990) have isolated, with an oligonucleotide probe corresponding to part of Phormia diptericin, a clone encoding a peptide structurally related to this antibacterial molecule. The deduced primary sequence of the Drosophila diptcricin shows an overall homology of approximately 60 % with that of the Phormia peptide with several regions (of 4 residues and more) of full homology (fig. 1). In particular, a pentagiycine stretch characteristic of the Phormia diptericins, is present in an equivalent relative position in Drosophila. Transcripts for the Drosophila diptericin are detected 2 h after injection of bacteria. The hybridization signal increases to its maximum at 4 h after which it becomes fainter. Interestingly, unchallenged early pupae and adults appear to contain low but significant amounts of diFtcricin transcripts which are however not detected in embryos or larvae (Wicker et al., 1990). In comparison to cecropin transcripts, the diptericin transcripts are found to be very abundant in immune ;ncpptc /l¢'vlctpn ot nl
IQOfi" W ; ~ l ~ r
ot
al., 1990). L ysozyme. Flyg and associates (1987) have detected lysozyme activity in Drosophila larvae and recently, Hultmark and coworkers have cloned the lysozyme locus in this species (Kylsten et al., personal communication). Functional lysozyme has not been detected in Phormia (unpublished results from this laboratory).
Expression of inducible antibacterial peptides The studies on Sarcophaga, Phormia and Drosophila conclusively point to the fat body as a major site of biosynthesis for the inducible antibacterial peptides. The role of the haemocytes is less well established, which is indeed not
surprising as they represent several cell lineages which are apparently not fully superposable in Drosophila and Phormia. We have recently compared in Phormia the expression of the predominant anti Gram-negative peptides in this species, i.e. the diptericins, and that of the major anti-Gram-positive molecules, the defensins (Dimarcq et al., 1990). The results show that all cells of the fat body express the genes coding for both peptides in a concomitant manner. In addition to the fat body cells, one single blood cell type, the thrombocytoids, also express concomitantly both types of genes. Thrombocytoids are remarkable blood cells in many respects, which undergo partial fragmentation during defence reactions, agglutinate around bacteria and seal off wounds of the integument. Interestingly, plasmatocytes which account for 80 % of the 10,000 to 12,000 cells per mm3 of blood of wandering larvae and are strongly phagocytic, do not express the genes ~or diptericins and defensins. Sarcophaga also contains thrombocytoids and, although this has not been specifically investigated, we assume that the undefined haemoc~es shown by ~.he Tokyo group to express defensin genes (sapecin) in Sarcophaga larvae correspond to this blood cell category (Matsuyama and Natori, 1988b). In contrast, Drosophila apparently lacks thrombocytoids. We did not observe expression of the diptericin gene in any Drosophila blood cell (unpublished), while Hultmark and associates monitored expression of the cecropin genes in some 10 % of the circulating haemocytes. These cells probably correspond to lamellocytes (Samakovlis et al., 1990). Studies based on transcriptional profiles have shown in Phormia that the concomitant expression of the genes encoding the anti-Gram-positive defensins and the anti-Gram-negative diptericins is independent from the nature of the stimulus: live or heat-killed Grampositive or Gram-negative bacteria, lipopolysaccharides, distilled water, induce the ev,~ression of both types of genes. Even a simple injury of axenical-
INSECT
ly raised larvae performed under strict sterile conditions is sufficient to induce the expression of diptericin and defensin genes in Phormia, suggesting that the disruption of the integument releases a signal or initiates a cascade of biochemical events, inducing the transcription of the genes encoding the antibacteria! peptides. When the response to a second stimulus was investigated in larvae which had been challenged by a first injection, it was found that the two responses were equivalent. This result does not argue in favour of a mechanism allowing the insect to keep a " m e m o r y " of the first injection (Dimarcq et al., 1990). In eonelusion, the humoral immune response of the three dipteran insects is primarily based on the rapid and transient transcription of a battery of genes encoding mostly small cationic antibacterial peptides. This response is a nonadaptative process which lacks both specificity and memory. The recent cloning of the genes encoding cecropins, diptericins and lysozyme of Drosophila shouid now enable *~-'~dissection of ,the control of the tran-
IMMUNITY
917
scription of these genes in this favourable biological model. In this context, the use of various cell lines should prove particularly rewarding. The signalling mechanism informing the adipocytes and blood cells of the injury may turn out to be more difficult to unravel, although some experimental models are now at hand. Of great interest are also the observations that some of the antibacterial peptides are synthesized during pupal development. These results point to a possible link between the endocrine system (control of metamorphosis) and the immune system in holometabolous insects (see contribution of Natori, this volume). As the antibacterial peptides of Diptera have now been either chemically synthesized (see Samakovlis et al., 1990 for Drosophila cecropins) or their cDNA expressed in heterologous systems (see Reichhart ar,d Achstetter, this volume, for Phormia defensin), the analysis of their mode of action and the structure-function relationship will be amenable to thorough investigations. Some of these molecules may eventually prove of interest in various fields of biotechnology (see Casteels, this Forum).
References.
ANDO, K. et ai. (1987), Purification of Sarcotoxin lI, antibacterial proteins of Sarcophaga peregrina (flesh fly) larvae. Biochemistry, 26, 226-230. ANDO,K. & NAa'ORI,S. (1988), Molecular cloning, sequencing and characterization of cDNA for Sarcotoxin IIA, an inducible antibacterial protein of Sercophagaperegrina (flesh fly). Biochemistry, 27, 1715-1721. BABA,K. et al. (1987), Purification of Sarcotoxin III, a new antibacterial protein of Sarcophaga peregrina. J. Biochem., 102, 69-74. BAKtJLA, M. (1970), Antibacterial compounds in the cell-free haemolymph of Drosophila melanogaster. J. Insect PhysioL, 16, 185-197. BOMAN, H.G. et al. (1972), Inducible antibacterial defence system in Drosophila. Nature (Lond.), 237, 232-234. DIMARCQ,J.L. et al. (1988), Insect Immunity. Purification and characterization of a family of novel inducible antibacterial proteins from immunized larvae of the dipteran Phormia terranovae and complete amino acid sequence of the predominant member, diptericin A. Europ. J. Biochem., 171, 17-22. D1MARCQ,J.L. et al. (1990), Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae. EMBO J., 9, 2507-2515. Er~csTgOM,A. et al. (1985), Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia. EMBO J., 4, 2119-2122. FLVC,C. et al. (1987), Insect Immunity. Inducible antibacterial activity in Drosophila. Insect Biochem., 17, 151-160.
918
34 th F O R U M I N I M M U N O L O G Y
GANZ, T. et al. (1990), Defensins. Europ. J. Haematol., 44, 1-8. HULTMARK,D. et al. (1980), Insect Immunity: purification and properties of thr~.e inducible bactericidal proteins from haemolymph of immunized pupae of Hyalophora cecropia. Europ. J. Biochem., 106, 7-16. HULTMARK,D. et al. (1982), Insect Immunity. Isolation and structure of cecropin D and four minor antibacterial components from cecropia pupae. Europ. J. Biochem., 127, 207-217~ HULTMARK,D. et al. (1983), Insect Immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J., 2, 571-576. KEpPI, E. et al. (1986), Induced antibacterial proteins in the haemolymph of Phormia terranovae (Diptera). Insect Biochem., 16, 395-402. KEPPh E. et al. (1989), Mode of action of diptericin A, a bactericidal peptide induced in the hemolymph of Phormia terranovae larvae. Arch. Insect Biochem. Physiol., 10, 229-239. KOCKUM,K. et al. (1984), Insect Immunity: isolation and ~equence of two cDNA clones corresponding to acidic and basic attacins of Hyalophora cecropia. EMBO J., 3, 207!-2075. KUZUHARA,T. et al. (1990), Determination of the disulfide array in Sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly). J. Biochem., 107, 514-518. KYLSTEN,P. et al. (1990), The cecropin locus in Drosophila, a compact gene cluster involved in the respt~nse to infection. EMBO J., 9, 217-224. LAMaEP.% J. et al. (1989), Insect Immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proc. nat. Acad. Sci. (Wash.), 86, 262-266. LEPAGE,P. et al. (1990), Determination of disulfide bridges in natural and recombinant insect defensin A. Europ. J. Biochem. (submitted). MATSUMOTO,hi. et al. (1986), Molecular cloning of cDNA and assignement of the C-termipal of Sarcotoxin IA, a potent antibacterial protein from Sarcophaga peregrina. Biochem. J., 239, 717-722. MATSUYAMA,K. & NATORI,S. (1988a), Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. J. biol. Chem., 263, 17112-17116. MATSUYAMA,K. & NATORI,S. 0988b), Molecular cloning of cDNA for Sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina. J. biol. Chem., 263, 17117-17121. OKADA,M. & NATORI,S. (1985a), Primary structure of Sarcotoxin I from Sarcophagaper~riha. J. biol. chem., 260, 7174~7177. OKADA,M. ~ NATORI,S. (1985b), Mode of action of a bactericidal protein induced in the haemolymph of Sarcophaga peregrina (flesh-tly) larvae. Biochem. J., 222, 119-124. POWN~NG,R.F. & DAWOSON,W.J. (1976), Studies on insect bacteriolytic enzymes. -- II. Some physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella. Comp. Biochem. Physiol., 55B, 221-228. REICHHART,J.M. et al. (1989), In~ect Immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera). Transcriptional profiles during immunization. Europ. J. Biochem., 182, 423-427. ROBERTSON,M. & POSTI.ETHWAIT,J.H. (1986), The humoral antibacterial response of Drosophila adults. Dev. Comp. Jmmunol., 10, 167-179. SAMAKOVLIS~C. et al. (1990), The immune response in Drosophila: pattern of cecropin expression and biological activity. EMBO J., 9, 2969-2976. STS~NER,H. et al. (1981), Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature (Lond.), 292, 246-248. VAN HOFSTEN,P. et al. (1985), Molecular cloning, cDNA sequencing and chemical synthesis of cecropin B from Hyalophora cecropia. Proc. nat. Acad. Sci. (Wash.), 82, 2240-2243. WICrER, C. et al. (1990), Insect immunity. Characterization of a Drosophila cDNA encoding a novel member of the diptericin family of immune peptides. J. biol. Chem. (in press).