Localization of the serine protease homolog BmSPH-1 in nodules of E. coli-injected Bombyx mori larvae and functional analysis of its role in nodule melanization.

The molecular mechanisms underlying nodule formation and melanization, an important pathogen defense mechanism in insects, are poorly understood. In this study, we investigated the role of BmSPH-1, a catalytically inactive Bombyx mori serine protease homolog, in nodule melanization induced by injection of Escherichia coli cells into the B. mori larval hemocoel. Addition of the melanization substrate l-3,4-dihydroxyphenylalanine (DOPA) to newly formed nodules prompted nodule melanization, confirming that nodules contain activated prophenoloxidase needed for melanization. Immunoprecipitation and immunoblot studies demonstrated that BmSPH-1 interacts with BmLBP, a C-type lectin that binds Gram-negative bacteria, and that BmSPH-1 is present in a truncated, putatively activated form at the E. coli cell surface in nodules. Pretreatment of larvae with anti-BmSPH-1 serum inhibited nodule melanization in E. coli-injected larvae. These results suggest that BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by BmLBP.