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.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

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.

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (Cys3 Cys30, Cysl6 Cys36 and Cys20 Cys38) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectrometry allowed the disulfide-bridge structure to be determined with a total of only 40 pg (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 pg recombinant insect defensin A, which is compatible with use in batch analysis.

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.