Protease inhibitors in Bombyx mori silk might participate in protecting the pupating larva from microbial infection

Abstract

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like (TIL)-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.

The fungal‐resistance factors BmSPI38 and BmSPI39 predominantly exist as tetramers, not monomers, in Bombyx mori

“…In addition, small amounts of trimer were also observed in some silkworm tissues, including the integument and haemolymph of the 3-day-old pupae. Furthermore, in our previous study, we found that large amounts of BmSPI39 could be secreted into the cocoon layer during the spinning process (Li et al, 2016b). In fact, BmSPI39 is quantitatively one of the most abundant protease inhibitors found in cocoon silk (Dong et al, 2013;Zhang et al, 2015;Guo et al, 2016).…”

“…However, the molecular masses of these trimeric and tetrameric forms of BmSPI39 were decreased dramatically at 108 h after the start of wandering (equal to the period of 2.5‐day‐old pupae). Notably, BmSPI39 protein also has a signal peptide of 24 amino acid residues (Li et al, ; ). Our data suggest that this N‐terminal signal peptide was removed from the pre‐mature BmSPI39 protein during the secretion process (Fig.…”

“…BmSPI39 has been shown to possess inhibitory potency against subtilisin A from B. licheniformis and protease K from E. album (Li et al, ) and can be secreted into the cocoon layer during spinning (Li et al, ). Therefore, we purified BSKIs using a protease K affinity column, and validated the activity of these inhibitors against subtilisin A from B. licheniformis and protease K from E. album using activity staining within a gel (Fig.…”

“…Western blot analysis was carried out as previously described with slight modification (Li et al, ; ). In this study, two different rabbit polyclonal antibodies directed against BmSPI38 or BmSPI39 (both diluted 1 : 2500) were used as the primary antibodies, while horseradish‐peroxidase‐labelled goat anti‐rabbit immunoglobulin G (IgG) antibody (diluted 1 : 40 000; Beyotime, Shanghai, China) was used as the secondary antibody.…”

“…Their antimicrobial activity has also been conferred for specific proteases, including subtilisin A from Bacillus licheniformis , protease K from Engyodontium album , protease from Aspergillus melleus and virulence protease CDEP‐1 of Beauveria bassiana (Li et al, ; ). Furthermore, both BmSPI38 and BmSPI39 have an inhibitory effect on B. bassiana conidia germination and could also be used as targeted resistance factors in medicine and agriculture (Li et al, ; ). However, the mechanism underlying the function of these TIL‐type protease inhibitors is largely unknown.…”