Serine protease inhibitor dipetalogastin-like from Galleria mellonella is involved in insect immunity

A new protein with immune properties was found in Galleria mellonella hemolymph. The so-far putative serine protease inhibitor dipetalogastin-like (GmSPID) was found in one fraction obtained after separation of hemolymph by RP-HPLC. Its amount depended on the immune status of the insect: it significantly increased after oral (10^3 CFU) and intrahemocelic (10 and 50 CFU) infection with entomopathogenic bacteria Pseudomonas entomophila. This was accompanied by up-regulation of the respective gene in the fat body of infected larvae. GmSPID was purified to homogeneity and characterised as a protein with immune properties. Among the three proteases tested, i.e. trypsin, elastase, and thermolysin, the strongest inhibition was observed toward trypsin. No inhibition toward the metalloproteinase thermolysin was detected, confirming that GmSPID is an inhibitor of serine proteases. Additionally, GmSPID was shown to have antimicrobial properties. At the concentration of 7 µM and 15 µM, it acted against Pseudomonas entomophila, Pseudomonas aeruginosa, Bacillus thuringiensis, Escherichia coli, and Candida albicans but not against Staphylococcus aureus. Moreover, with the use of atomic force, scanning, and transmission electron microscopy techniques, we present the effect of the GmSPID protein on the surface properties, shape, and ultrastructure of P. entomophila cells. The protein caused modest perforation of the cellular membrane, contributing to loss of its integrity. The mode of the GmSPID protein action as an antimicrobial compound and its role in G. mellonella immunity are discussed.