A prophylactic role of a secretory PLA2 of Spodoptera exigua against entomopathogens

Phospholipase A(2) (PLA(2)) hydrolyses phospholipids at sn-2 position to release free fatty acids and lysophospholipids. Secretory type of PLA(2) (sPLA(2)) has been found in many different animals including insects. Insect sPLA(2)s have been divided into venomous and nonvenomous PLA(2)s. A non-venomous sPLA(2) (Se-sPLA(2)) has been identified in beet armyworm, Spodoptera exigua. Its high enzyme activity is detected in hemolymph of naive larvae. However, the physiological role of high sPLA(2) activity in hemolymph remains unclear. To determine the physiological role of sPLA(2) in hemolymph, a recombinant Se-sPLA(2) (rSe-sPLA(2)) was expressed in a bacterial expression system and purified to test antimicrobial activity against various microbes. Purified rSe-sPLA(2), exhibited typical enzyme kinetic properties, including becoming saturated at high substrate concentrations, exhibiting optimal activity at pH 7-9, and being inactivated at high temperatures. However, a reducing agent (dithiothreitol) or calcium chelator treatment inhibited the catalytic activity. A specific inhibitor to sPLA(2) also inhibited the enzyme activity of rSe-sPLA(2) while other type PLA(2) inhibitors did not. Furthermore, eight bacterial metabolites of Xenorhabdus and Photorhabdus known to be inhibitory against insect PLA(2) significantly inhibited the enzyme activity of rSe-sPLA(2). High concentrations of rSe-sPLA(2) (above 0.5 mM) showed significant cytotoxicity to hemocytes of S. exigua. At concentrations without showing cytotoxicity, rSe-sPLA(2) possessed significant antimicrobial activities against entomopathogenic bacteria (Serratia marscens and Entercoccus mondtii) and fungi (Beauveria bassiana and Metarhyzium rileyi). Hemolymph obtained from larvae treated with RNA interference specific to Se-sPLA(2) significantly lost such antimicrobial activities. However, the addition of rSe-sPLA(2) to the hemolymph significantly rescued such antimicrobial activities. These results indicate that Se-sPLA(2) possesses antimicrobial activity, suggesting that it might act as a prophylactic agent against microbial pathogens in the hemolymph of S. exigua.