We have isolated from the hemolymph of immunized larvae of the dipteran insect Phormia terranovae two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues containing three intramolecular disulfide bridges and differ from one another by only a single amino acid. These peptides are neither functionally nor structurally related to any known insect immune response peptides but show significant homology to microbicidal cationic peptides from mammalian granulocytes (defensins). We propose the name "insect defensins" for these insect antibiotic peptides.

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat-stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N-terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.It is now well established that lepidopteran and dipteran insects respond to a bacterial challenge and also to injury by synthesizing peptides with antibacterial activity (reviewed in Boman and Gotz [l]). Several antibacterial molecules induced in Hyalophora cecropia have been fully or partially characterized : these are the cecropins (3 -5 kDa basic, heatstable peptides) [2], the attacins (20 kDa basic or acidic proteins) [3] and lysozymes [4]. In dipterans, only one induced antibacterial protein has been characterized so far; it is a basic 39-residue molecule named sarcotoxin I, isolated from the blood of injured flesh-fly larvae Sarcophaga peregrina; this molecule shares significant homology with cecropins and is found in at least three forms [5].In the course of an investigation into the cellular and humoral defence reactions of the dipteran Phormia terranovae, a species closely related to Calliphora erythrocephala, we have recently obtained evidence for the appearance in the haemolymph of immunized larvae of a number of heat-stable, basic antibacterial proteins [6, 71. There are at least five of these proteins (or groups of proteins) in the immune haemolymph; none of them corresponds to lysozyme.In the present paper we show that three of these proteins belong to a novel class of antibacterial peptides; in particular, they differ from cecropins, attacins and lysozymes. We propose that they should be named diptericins. We report how, starting with immune plasma of Phormia, we have isolated the induced protein which shows the highest antibacterial activity on Escherichia coli in our assay conditions. It is a basic molecule (PI = 8.5) containing 82 amino acid residues with a relative molecular mass of 8610. We have determined the complete amino acid sequence of this major form (diptericin A) and sufficient N-terminal sequence of two of the minor forms (diptericins B and C) to reveal that there is a family of diptericins of related structure.

A bacterial challenge of larvae of the dipteran insect Phormia terranovae induces the rapid synthesis of diptericin, an antibacterial polypeptide, previously characterized at the amino acid level and indirectly by cDNA cloning studies. This 82-residue polypeptide consists of an N-terminal proline-rich domain and a central and C-terminal glycine-rich domain. Using liquid chromatography coupled to electrospray ionization-mass spectrometry, we demonstrate here that this molecule is more complex than anticipated and carries two O-substitutions on threonine residues, one in the proline-rich domain (residue 10) and one in the glycine-rich domain (residue 54). These substitutions consist of identical trisaccharides: glucose-->galactose-->N-acetylgalactosamine-->(threonine). Treatment of diptericin with O-glycosidase, which selectively removes the substitutions without altering the polypeptide proper, abolishes the antibacterial activity, indicating that this posttranslational modification is essential for biological activity of the polypeptide. We also show that diptericin is posttranslationally modified by a C-terminal amidation.

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Diptericin A is a member o f a multigenic family of antibacterial peptides that are synthesized by larvae of fhormia terranovae (Diptera) in response to a bacterial injection o r to injury. The 82-residue peptide is active only against a limited range o f Gram-negative bacteria. Data presented suggest that the primary action of diptericin A is on the cytoplasmic membrane of growing bacteria.