Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(l-13)-melittin(l-13) was IOO-fold more active than cecropin A against Staphylococcus uureus. It was also a lo-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.Cecropin A; Melittin; (Staphylococcus aureus, Plasmodium falciparium)

Two cDNA clones containing coding information for cecropin B from the Cecropia moth (Hyalophora cecropia) were identified by means of a synthetic probe. Sequencing of the two inserts showed that cecropin B is processed from a 62-amino acid residue precursor molecule including a 26-residue leader peptide and a COOH-terminal glycine residue. The latter presumably donates the nitrogen of the amide group present on the COOH-terminal leucine residue of the mature cecropin B. The sequence deduced for the mature cecropin B differed in the COOH-terminal region from the tentative structure previously determined by carboxypeptidase digestion. To settle the discrepancy, cecropin B was syntjeesized according to the cDNA sequence with an amidated COOH-terminal leucine. Natural and synthetic cecropin B were found to be indistinguishable with respect to electrophoretic mobility and antibacterial activity against seven different bacteria. The COOH-terminal tetrapeptides were isolated from both natural and synthetic cecropin B and found to be indistinguishable. The correct sequence for cecropin B is LysTrp-Lys-Val-Phe-Lys-Lys-Ile-Glu-Lys-Met-Gly-ArgAsn-Ile-Arg-Asn-Gly-Ile-Val-Lys-Ala-Gly-Pro-AlaIle-Ala-Val-Leu-Gly-Glu-Ala-Lys-Ala-Leu-NH2.Humoral immunity can be induced in many insects by an injection of live nonpathogenic bacteria. This phenomenon has been analyzed at the molecular level by using diapausing pupae of the Cecropia moth (Hyalophora cecropia) as a model system (1, 2). After a short period of RNA synthesis the insects respond to the bacteria by the production of a potent antibacterial activity which is due to the composite action of lysozyme and two new classes of bactericidal proteins, the cecropins (3, 4) and the attacins (5-7). The cecropins are small basic proteins with a comparatively long hydrophobic region. The three principal cecropins, A, B, and D, were isolated from immune Cecropia hemolymph. Sequence work revealed a high degree of homology among these three forms, which suggests that they have evolved through gene duplications. Similar conclusions were reached concerning the two main forms of attacin.To understand the selective induction of the immune genes, a gene cloning program has been initiated. cDNA clones and sequences for the two main forms of attacin were first obtained (7,8). We now report the isolation and sequence of two cDNA clones together containing the complete information for the precursor of cecropin B. The amino acid sequence deduced for the mature form of cecropin B differs at two residues in the COOH terminus from the tentative structure previously obtained from sequencing the peptide (3). To settle the discrepancy we synthesized the peptide predicted from the DNA sequence. A detailed comparison of the natural and the synthetic materials indicates that the two products are identical.

We have previously shown that pupae of the giant silkmoth Samia cynthia have a humoral antibacterial activity, which was induced by viable, nonpathogenic gram-negative bacteria (H. G. Boman et al., 1974). We show here that this activity was formed simultaneously with a selective incorporation of amino acids into eight polypeptide chains characterized by their electrophoretic behavior. If actinomycin D or cycloheximide were given at an early time, no antibacterial activity was found. If the inhibitors were given at the time of maximum activity, there was no effect with actinomycin D but a rapid decrease of the activity in the case of cycloheximide. The results imply that the messenger ribonucleic acid was stable, but that at least one protein component was turning over. Hemolymph from immunized pupae of another giant silkmoth, Hyalophora cecropia, was fractionated by ammonium sulfate precipitation. This procedure, together with the isotope distribution after co-electrophoresis in polyacrylamide gels, was used for comparing the response to injury and to different infections. Almost identical polypeptide patterns were obtained as a response to an infection with either viable Enterobacter cloacae or Bacillus subtilis. These patterns differed both qualitatively and quantitatively from the injury effect created by an injection as such. There was only a low antibacterial activity in each of the four fractions obtained by ammonium sulfate precipitation. However, a combination of three fractions restored a high killing activity. Fractionation of hemolymph from untreated pupae provided evidence for at least one preexisting factor which stimulated the killing of Escherichia coli. The osmotic pressure of the bacteria contributed to the antibacterial activity towards E. coli, but not towards B. subtilis. The killing of E. coli was inhibited by lipid A and, to a lesser extent, by an inhibitor of proteolytic enzymes. The similarities and differences with the mammalian complement system are discussed.

Cecropin B and cecropin IA (sarcotoxin IA) are 35‐ and 39‐residue antibacterial peptides from a silk moth and a meat fly, respectively. Using solid phase synthesis we have made these peptides as well as two 37‐residue analogs, one containing a deletion of leucine and lysine (residues 2a and 2b) as compared to cecropin IA, the other containing an insertion of leusine and lysine at the corresponding place in cecropin B. This addition and removal of a lysine residue did not cause the expected change in electrophoretic mobility. When tested for antibacterial spectra, the insertion analog was found to be as active as the parent compound while the deletion analog had lost most of its antibacterial capacity. In addition it was shown that the C‐terminal amide contributes to the broad spectrum properties of the cecropins.

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