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Development of Novel Insecticidal Compounds Using Eicosanoid Biosynthesis Inhibitors Originated from Entomopathogenic Bacteria
An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression by inhibiting phospholipase A2 (PLA2), which results in a fatal septicemia. PLA2 is an enzyme responsible for eicosanoid biosynthesis and the pathogenic molecular target of this bacterium. A PLA2 gene of the red flour beetle, Tribolium castaneum, was expressed in Escherichia coli. The recombinant T. castaneum PLA2 (TcPLA2) showed enzyme activity, which was specifically inhibited by bromophenacyl bromide (specific inhibitor to secretory PLA2) and ditheothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 7.0), temperature (optimum at 30°C), substrate specificity and calcium dependency. X. nematophila released compound(s) inhibiting TcPLA2during its stationary growth phase. The active compound (s) was resistant to heat treatment and could be extracted separately into both organic and aqueous phases. This PLA2 inhibitory fraction showed significant effect on immunosuppression of T. castaneum. These results suggest there may be several PLA2 inhibitors synthesized by X. nematophila and released into culture broth. The recombinant TcPLA2 was also used to screen potent PLA2 inhibitor compounds, which were designed based on a common chemical structure (a pentenebenzene ring) of two peptide inhibitors, proline-tyrosine (PY) and acetylated phenylalanine-glycine-valine (AcFGV). Alterations were made on amino acid sequence or specific functional groups on the pentenebenzene ring. Among 7 different peptides, AY and FGV showed the most potent effects on TcPLA2activity and also resulted in significant reductions in hemocyte spreading behavior of Plutella xylostella. The potent candidate molecules would be applied to control various insect pests to be developed into novel insecticides.
