Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa.

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.