An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A(2) (PLA(2)) activity. Recently, an immune-associated PLA(2) gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA(2) gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA(2) (TcPLA(2)) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA(2) (sPLA(2)) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA(2) inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA(2)). The X. nematophila culture broth contained PLA(2) inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA(2) inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA(2)-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA(2) gene expression by RNA interference.
