INSECT IMMUNITY

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EXPRESSION A N D SECRETION O F INSECT IMMUNE PEPTIDES IN YEAST

J.-M. Reichhart Q) and T. Achstetter (2)

(l) Laboratoire de Biologie Gdn6rale de I'Universit~ Louis Pasteur, S/rasbourg, and (2) Transgbne, Strasbourg (France)

A major drawback of studies on the mode of action, the antibacterial activity spectrum and the tridimensional structure of insect immune peptJdes is the small amount of material which can be extracted from the respective biological sources. The paucity of native material is also a handicap in exploring the potential o f these molecules as novel antibiotics. For the r~=~ativelysmall cecropins (37 and 39 residues) which lack cysteins and hence d~sulphide bridges, chemical synthe~E appeared to be a straightforward answer to this problem (Boman et al., 1989). However, for complex molecules, like defensins, which have three intrachain disulphide bridges within their 40-residue sequence, as well as for larger peptides, like diptericins (82 residues) and attacins (270 residues), chemical synthesis daes not appear to be a method of first choice. We have explored with our coworkers (Reichhart et aL, submitted) the feasibility of expxesdng immune peptide genes from insects (specifically from Phormia terranovae and f r o m Drosophila melanogaster) in a heterologous system, namely in yeast. When we i.,,itiated this project 18 m~,nths ago, no insect peptide gene had been expressed in yeast, at least to our knowledge. We therefore relied on one of the established expression and secretion systems of yeast, in which the coding sequence of the gene to be expressed is placed in frame with the prepro-sequet~ce of the yeast pheromone mating factor a (MFal) gene. The MFal gene encodes a precursor of the a mating factor and during the export of this precursor, the pro-region is cleaved off by the yscF endoprotease

(KEX2 gene product) probably in a trans-Golgi compartment. This property has been utilized to direct the release of several correctly matured polypeptides into the medium, namely that o f hirudin (Loison et aL, 1988; see also references therein). The expression cassette consisted, in our first experiments, o f the promoter of the MFal gene, the prepro-sequence of this factor, the coding sequence of the immune peptide gene, and was followed by the phosphoglycerate kinase gene (PGK) terminator. The expression cassette was introduced into a lnulticopy yeast-Escherichia coil shuttle vector containing the origins of replication for yeast (2-t~ p l a s m i d ) aiid E~ coil (pBR322) and the selection markers for uracil and leucine prototrophy and ampicillin resistance, respectively (fig. 1). Using the preferential codon choice of yeast, we have first constructed a synthetic gene corresponding to the coding sequence of insect defensin A (Lambert et al., 1989; see Hoffmann and Hoffmann, this volume). The gene was constructed to allow, through appropriate restriction sites, in-frame cloning into the expression cassette. After transformation by the vector containing the synthetic defensin-coding sequence, yeast clones were grown under selective conditions on agar plates and the potential secretion of the insect defensin A peptide was monitored by a growth inhibition assay ~sing Micrococcus iuteus as test organism, as shown in figure 2. Controls were run with yeast transformed with a vector lacking only the defensin-coding sequence. It is evident from figure 2 that yeast clones expressing defensin A do secrete into the medium molecules active aga:inst M. luteus.

944

34 th F O R U M I N I M P A U N O L O G Y were identical in both types of molecules (Lepage et al., 1990).

Quantification of the recombinant antibacterial molecules (liquid growth inhibition assay calibrated with authentic defensin A) indicated that 0.5 ~tg defensin equivalents are accumulated in 1 ml of culture medium. Under appropriate large scale fermentation conditions with a yeast strain harbouring the expressiot~ vector, yields of 60 mg/l are now routinely obtained. The recombinant insect defensin was purified and subjected to peptide sequencing by Edman degradation. The amino acid sequence was found to be identical to the authentic molecule, indicating that the precursor peptide had been correctly processed. Liquid secondary ion mass spectrometry showed that the molecular mass of the recombinant defensin was the same as that of the native molecule and a cletailed mass spectrometry analysis confirmed that the positions of the three disulphide bridges

Given that these results represented, to our knowledge, the first successful expression of a biologically active insect peptide in yeast, we undertook a series of constructions in which we replaced the synthetic defensin gene (yeast codon choice) by the Phormia nucleotide sequence, and the pro-sequence of M F a l by the Phormia defensin precursor prosequence (Dimarcq et al., 1990). The expression of active defensin was obtained and the yields were similar to those observed in the first experiments described above. These results indicate that the yeast translation machinery can correctly deal with an insect-derived nucleotide sequence. We have also tried to express the diptericin gene in yeast, using the same methodological approach (an expres-

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FIG. 1. - - Structure ofpTG3856. This vector is based on plasmid pTG881 (Loison et aL, 1989) which is an E. coli/yeast shuttle vector designed to direct the secretion of foreign proteins in yeast. It contains the URA3 and LEU2-d selection markers with a fragment of the 2-micron plasmid as well as the Ap a gene and the origin of replication of pBR322. This plasmid was modified by introduction of silent mutations encoding a unique HindlII site 15 nucleotides upstream of the first amino acid codon of the ct factor yielding pTG 1895 (Achstetter et aL, in preparation). To obtain pTG3856, the HindllI-BgllI fragment of pTGI895, containing the a factor sequence was replaced by the synthetic defensin A gene as a HindllI-BamHl fragment. PROM = MF~I promoter. Pre~pro = MFal pre- and pro-sequences. TERM = PGK terminator,

INSECT IMMUNITY

945

~ ~ i ! : i ~¸I~~I : i ! i¸:~ ~ : :iii¸¸¸ ::i¸~I~I:~!~: / ..... FIG. 2. --Expression o f anti-M, luteus peptides by yeast cells. Transformed yeast cells were plated on minimal agar containing medium at pH 6.8 and incubated for 48 h at 30°C. They were overlain with 4 ml of top-agarose containing log phase cells of M. luteus (0,80D6o o nmper ml) and incubated overnight at 37°C. Note growth inhibition zones around yeast clones transformed with the insect defensin A containing construct (pTG3856: right half) versus controls lacking the defensin-codingsequence (pTG1895: left half).

sion cassette consisting of the promoter and prepro-sequence of the M F a l gene fused to the diptericin cDNA sequence from Phormia (Reichhart et al., 1989) and the P G K terminator inserted in the same type of muiticopy shuttle vector). Several C-terminally truncated diptericins were produced under these conditions which exhibited only reduced biological activity. Phormia diptericin is C-amidated (Reichhart et aL, 1989) and it is presently unknown whether yeast is capable of carrying out this post-translational modification. It is an open possibility that the lack of Camidation renders the newly synthesized

diptericin molecules more susceptible to C-terminal degradation. We are now exploring the expression of Drosophila diptericin, the cDNA of which has been recently cloned and which has no amidated C-terminus (Wicker et al., 1990). Our results concerning defensin A from Phormia provide a first encouraging example for an efficient expression of an insect gene in yeast. They also have the advantage of making available reasonable amounts (in the 100-mg range) for studies on the structureactivity relationship of this molecule and for exploring its potential role as a novel antibiotic.

946

34 th F O R U M I N I M M U N O L O G Y

References. BOMAN,H.G., BOMAN,A., ANDREU,D., LJ, Z.Q., MERRIFIELD,R.B., SCHLENSTEDT,G. & ZIMMERMANN,R. 0989), Chemical synthesxs and enzymic processing of precursor forms of cecropins A and B. J. biol. Ct~em., 264, 5852-5860. D1MARCQ,J.L., ZACHARY,D., HOFFMANN,J.A., HOFFMANN,D. ~; REICHHART,J.M. (1990), Insect immunity: expression of the two major inducible antibacterial peptides, defensin and diptericin, in Phormia terranovae. EMBO J., 9, 2507-2515. LAMBERT,J., KEPFI,E., DIMARCQ~J.L., WICKER,C., REICHHART,J.M., DUNBAR,B., LEFAGE~ P., VANDORSSELAER,A., HOFFMANN,J., FOTHERGILL,J. & HOFFMANN,D. (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., BITSCH,F., ROECKLIN,D., KEPPI,E., DIMARCQ,J.L., REICHHART,J.M., HOFFMANN, J.A., ROITSCH,C. d~ VANDORSSELAER,A. (1990), Determination of disulfide bridges in natural and recombinant insect defensin A. Europ. J. Biochem. (in press). LolsoN, G., FmDELbA., BERNARD,S., NGUVEN-JutLLERET,M., MARQUET,M., EIE,L-BEELON,N., CARVALLO,D., GUVRRA-SANTOS,L., BROWN,S.W., COURTNEV,M., ROtTSCH,C. & LEMOmE, Y. (1988), Expression and secretion in Saccharomyces cerevisiae of biologically active leech hirudin. Biotechnology. 6, 72-77. LolsoN, G., VIDAL,A., FINOELI,A., Rolvsc8, C., BALLOUL,J.M. & LEMOINE,Y. (1989), High level of exp~'ession of a protective antigen of Schistosomes in Saccharomyces cerevisiae. Yeast, 5, 497-507. REICr~RT, J.M., ESSRiCH,M., DIMARCQ,J.L., HOFFMANN,D., HOFFMANN,J.A. & LAGUEUX, M. (1989), Insect immunity. Isolation of eDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera). Transcriptional profiles during immunization. Europ. J. Biochem, 182, 423-427. REICHHART,J.M., LEGRAIN,M., KEPPI,E., PETIT,I., DIMARCQ,J.L., LECOCQ,J.P., HOFFMANN, J.A. & ACHSTETTER,T., Expression and secretion in yeast of active insect defensin, an inducible antibacterial peptide from Phormia terranovae (Diptera) (submitted for publication). WICKER,C., REICHHART,J.M., HOFFMANN,D., HULTMARK,D., SAMAKOVLIS,C. ~ HOFFMANN, J.A. (1990), Insect immunity. Molecular cloning of a Drosophila eDNA encoding a novel member of the diptericin family of immune peptides. J. biol. Chem. (in press).

DISCUSSION

K. S6derh~ill, A. Aspdn and B. Duvic: Is the proPO-system involved in defence and recognition in arthropods ? That the proPO-system appears to function as a recognition system of arthropods has been suggested mainly because microbial polysaccharides such as fungal [3-1,3-glucans and bacterial LPS could induce activation of the proPOsystem. Recent data from work on crustaceans provide more solid support that the proPO-system appears to be a defence and recognition system. In crustaceans, the different haemocyte types are more easy to separate and to handle in vitro than those of insects and, as a consequence, it has been pos-

sible to study how different proPOproteins interact with the different haemocyte types. Thus, a 76-kDa factor, whose biological activity is generated concomitant with the activation of the proPO-system, has been shown to have multifunctional properties, since it can mediate cell adhesion, degranulate semigranular and granular blood cells and finally function as an encapsulating promoting factor or as an opsonon-like substance. So, at least in crustaceans a molecule is present which is directly involved in the communication between blood cells. Since the proPO-system and thus also the 76-kDa protein is localized in the blood cells and has to be released from these cells by a regulated exocytosis to exert its function, one would