Insect immunity. Isolation of cDNA clones corresponding to diptericin, an inducible antibacterial peptide from Phormia terranovae (Diptera)

Abstract

We have previously isolated and characterized a family of novel 8-kDa cationic antibacterial peptides synthesized by larvae of Phorrnia terranovae (Diptera) in response to various injuries. These molecules have been named diptericins. The peptide sequence of diptericin A was used to prepare oligonucleotides for screening cDNA libraries and we report in the present paper the isolation of several cDNA clones encoding diptericin. The analysis of the nucleotide sequences indicates that diptericin is synthesized as a prepeptide which is matured in two steps: (a) cleavage of a signal peptide and (b) amidation of the C-terminal residue. Interestingly, the 3' untranslated region of the mRNA contains a consensus sequence TTATTTAT which is also observed in the mRNA of another insect antibacterial peptide (attacin-related sarcotoxin IIA) and in mRNAs encoding proteins related to the inflammatory response in mammals. Our data illustrate that diptericins form a polymorphic family of immune peptides. The transcription of the diptericin genes is rapidly induced in the fat body after inoculation of bacteria, as evidenced by the transcriptional profile.Our knowledge of the biochemical aspects of the immune response in insects has developed significantly over the last decade. It has been clearly established that in the higher insect orders, namely Lepidoptera and Diptera, injection of live or killed bacteria and even a simple injury, induce the rapid appearance in the haemolymph of several families of antibacterial peptides (reviewed in [I -31). We have recently isolated from the immune haemolymph of larvae of the dipteran Pliormiu terranovae three heat-stable peptides active on Gramnegative bacteria. We have established the full amino acid sequence of one of these molecules which contains 82 residues and has a relative molecular mass of 8610. The two other peptides have only been partially sequenced (the first 40 residues from the N-terminus) and exhibit marked similarities, indicating that the three peptides belong to a common family [4]. They are not structurally related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxins 1) and attacins (including sarcotoxins II)] and have no significant similarity with other reported peptide sequences. These novel antibiotic molecules have been named diptericins (diptericin A referring to the fully sequenced peptide [4]). The present paper reports the isolation of several cDNA clones for diptericins from which the structure of the diptericin precursor molecule is deduced. In addition, some of these clones served to investigate the transcriptional profiles for the diptericin genes during immunization. This infor- mation is one of the prerequisites for the analysis of the mechanism of selective induction of the immune genes, a question which will be addressed in a subsequent study. MATERIALS AND METHODS InsectsThird instar wandering larvae (crop half empty), 2-day-old pupae and 1-week-old male and female adults of P. terrnnovae were isolated and kept...

Insect antimicrobial peptides and their applications

“…Both tenecin-4 and hemiptericin share ~22% identity to H. gloveri gloverin, but tenecin-4 has higher identity (22–24%) to H. virescens and T. ni gloverins than hemiptericin (15–17% identity). Other glycine-rich peptides are 7–10 kDa, such as P. terranovae and D. melanogaster diptericins (82 residues) (Dimarcq et al, 1988; Reichhart et al, 1989; Wicker et al, 1990), S. peregrina sarcotoxin-III (7 kDa) (Baba et al, 1987), Zophobas atratus and Allomyrina dichotoma coleoptericins (72–74 residues) (Bulet et al, 1991; Sagisaka et al, 2001), H. diomphalia holotricin-2 (72 residues) (Lee et al, 1994), Acalolepta luxuriosa acaloleptins (~8 kDa) (Imamura et al, 1999), and honeybee hymenoptaecin (93 residues) (Casteels et al, 1993). These glycine-rich peptides have activities against Gram-negative and Gram-positive bacteria.…”

The humoral antibacterial response of Drosophila

“…We used an oligonucleotide probe corresponding to the peptide sequence of the newly-isolated diptericin from the fleshfly Phormia terranovae [22]. A fourth cecropin gene (C), encoding a peptide which differs from cecropin A by three conservative amino-acid replacements, was later described [23].…”

Determination of disulfide bridges in natural and recombinant insect defensin A

“…Furthermore, the measured mass compared to the predicted mass (4060.6 Da) from the amino acid sequence, eliminated any possibility of post-translational modifications involving variations of more than 1 Da. A monoisotopic mass measurement (measured, 4057.90 Da; expected, 4057.81 Da) with an accuracy better than 0.1 Da was also achieved [l] and it was thus possible to demonstrate that the C-terminus was not amidated (expected mass difference of -1 Da) although this has been described for other antibacterial peptides like cecropin isolated from Hyalophora cecropia [2] and the related sarcotoxin IA isolated from Sarcophaga peregrina [3], as well as diptericin isolated from P. terranovae [4]. Finally, the data obtained from mass spectrometry measurements also showed that natural insect defensin A was not present in a covalent dimeric form since the molecular ion did not exhibit peaks separated by mjz 0.5, and no peak was detected at mjz 8120 [l].…”

“…The LSIMS spectrum for both peptides presents the protonated molecular ion at mjz 889. 4 and two peaks at m/z 401.2 and 491.2, corresponding to the protonated molec-- Table 1. Measured m/z of the major peaks from Fig.…”