Screening of insect immune suppressors using a recombinant phospholipase A2 of a lepidopteran insect

Abstract

Phospholipase A₂ (PLA₂) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA₂ catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA₂ genes (As-PLA₂A–As-PLA₂D), in which As-PLA₂A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA₂A expression significantly reduced the PLA₂ activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA₂A protein was purified from a bacterial expression system, which exhibited a typical Michaelis—Menten kinetics and hence susceptible to a specific inhibitor to sPLA₂ and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA₂A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA₂A protein can be applied for high-throughput screening of insect immunosuppressive compounds.