Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity

Abstract

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host–pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 μM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.