Identification And Biological Activities Of Antimicrobial Proteins In Bombyx Mori Cocoon Silk

Silkworm is the model of Lepidopteran insects, which needs undergoes complete metamorphosis to become adult from larvae, and the pupal stage is an indispensable stage for silkworm. During the pupal stage, silkworm cannot move but have to face natural enemies, including a variety of predators and pathogenic microorganisms. To survive in such hostile environment, silkworm make cocoons to protect them after the long-term evolution. It is also because of the cocoon that makes the silkworm become an important insect with great economic value.B. mori cocoon is mainly composed of fibroins and sericins, which form silk fiber with prominent physical properties, and can provide perfect protection for the silkworm. In addition, a variety of other protein in the cocoon may also protect pupa from the invasion of pathogenic microorganisms by biochemical means. In order to prove whether the proteins in the cocoon can inhibit the growth of microorganisms, and discover the antimicrobial components, we groped and built the suitable conditions for the antimicrobial experiments against E.coli and Beauveria bassiana. On this basis, we found that cocoon proteins can obviously inhibit the germination of B. bassiana spores. By activity staining and activity assay, we found that the inhibitor activity is associated with antifugal activity. The Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used and revealed that the active components in the silk are protease inhibitors. The main results are as follows:1. The exploration and establishment of conditions for the antimicrobial experiments of cocoon proteins against bacteria and fungusE. coli were selected as one of experimental objects. We established a method to extract the active silk proteins, and determined the inhibition of silk proteins with different quality against E. coli with different concentrationsby disk diffusion method, cup plate method, OD measured and the cultivating method. All results showed that cocoon proteins can not inhibit the growth of E. coli. This result is same as that in a literature before.One of the main pathogenic microorganism, B. bassiana was then selected as the experimental object. The spore incubation method were established for experiment. The best experimental conditions is 26℃,80 rpm, the most suitable observation time is 16 h and the best quality of silk proteins is 150 μg. We found that cocoon proteins can obviously inhibit the germination of B. bassiana spores under the most suitable experimental conditions. Then, nystatin, a kind of broad-spectrum antifungal drugs, was used as the positive control under the most suitable experimental conditions. We found that the effect of 150 μg silk proteins was equivalent to that of 0.3～0.5 μg nystatin.2. Identification of the antifugal components in the cocoon silkFirst, we divided the cocoon into six layers and extracted the water-soluble proteins from the cocoon, and found that they have a strong inhibitory activity against fungal protease and showed an increase trend from inner cocoon layers to outer cocoon layers. Moreover, we found that they can obviously inhibit the germination of B. bassiana spores. Consistent with the changing trend of the protease inhibitors, we found that proteins in the outermost cocoon layer showed better inhibitory effect against the B. bassiana spores than that in other cocoon layers. The protease inhibitors showed unbelievable stability, remaining have inhibitory activity against fungal protease and germination of B. bassiana spores even after being boiled. It is because that protease inhibitors in the cocoon contain many intramolecular disulfide bonds. To reveal the active components in the silk, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used and identified 129 proteins. The label-free quantification indicated that 30 protease inhibitors accounted for 89.1% of the total protein abundance. In boiled scaffold proteins, inhibitors also showed the highest molar abundance of 93.4%, but in the precipitate, enzyme showed the highest molar abundance of 65.1%. Inhibitors in scaffold proteins and boiled scaffold proteins were analyzed. The highest molar abundance of inhibitors was kunitz-type serine protease inhibitor BmSPI51, it was found acccounted for 67% and 66% in the scaffold and boiled scaffold, respectively. The TIL-type serine protease inhibitor BmSPI39 had the second highest molar abundance, accounting for 22% and 25% of the total abundance in scaffold and boiled scaffold, respectively. Other inhibitors only made up of about 11% and 9% of the total molar abundance. These results can proved that inhibitors in cocoon plays major roles to inhibitthe germination of B. bassiana spores.3. Determination of antimicrobial acitivity of high-abundance protease inhibitors in the cocoonIn order to verify the above conclusions, we isolated BmSPI51 from scaffold proteins with natural purification. It showed weak activity by activity test. The protease inhibitors BmSPI38 and BmSPI39 were obtained by prokaryotic expression. BmSPI38, BmSPI39 and BmSPI51 was then used for the spores incubation experiment, respectively. It is showed that BmSPI51 had weak effect on B. bassiana spores, but BmSPI38 and BmSPI39 can obviously inhibit the germination of B. bassiana conidia. These results proved that protease inhibitors in the cocoon play important roles to inhibit the germination of B. bassiana spores.