Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade

Nosema bombycis is a pathogen of the silkworm that belongs to the microsporidia, a group of obligate intracellular parasites related to fungi. N. bombycis infection causes the disease pebrine in silkworms. Insects utilize hemolymph melanization as part of the innate immune response to fight against pathogens, and melanization relies on a serine protease-mediated prophenoloxidase (PPO) activation cascade that is tightly regulated by serine protease inhibitors (serpins). Previous studies showed that N. bombycis infection suppressed silkworm hemolymph melanization, however the mechanism has not been elucidated. We hypothesize that N. bombycis can secret serpins (NbSPNs) to inhibit host serine proteases in the PPO activation cascade, thus suppressing phenoloxidase (PO) activity and the consequent melanization. We demonstrated in this study that N. bombycis infection suppressed silkworm PO activity and melanization and we identified the expression of N. bombycis serpin 6 (NbSPN6) in the hemolymph of the infected host. When recombinant NbSPN6 was added to normal hemolymph, PO activity was inhibited in a dose-dependent manner. Moreover, in vivo analysis by RNA interference technology showed that when NbSPN6 expression is blocked, the inhibitory effects on PO activity can be reversed and the proliferation of N. bombycis within host can be suppressed. These results demonstrated the indispensable role of NbSPN6 in successful pathogen infection. To further elucidate the molecular basis of NbSPN6 suppressing host defense, we determined that the host serine protease prophenoloxidase-activating enzyme (PPAE) is the direct target of NbSPN6 inhibition. Taken together, our novel study is the first to elucidate the molecular mechanism of pathogen-derived serpin inhibiting hemolymph melanization and, thus, regulating host innate immune responses. This study may also provide novel strategies for preventing microsporidia infection.