The Molecular Mechanism Underlying Resistance Against Entomopathogenic Fungi By Protease Inhibitors In Bombyx Mori

Bombyx mori is a silk-producing insect with tremendous economic value. Based on the immense accumulation of fundamental research, B. mori has gradually developed into a model organism of Lepidoptera. However, silkworm is subject to environmental threats of various pathogenic microorganisms and parasites, which lead to serious diseases. Entomopathogenic fungi such as Metarhizium anisopliae, Beauveria bassiana, Aspergillus flavus, and Aspergillus oryzae can cause various communicable diseases, which seriously affect the production and quality of cocoon, and cause enormous economic losses in the silkworm industry. Entomopathogenic fungi, as a new biopesticide, has been widely used in agriculture and forestry pest control and mosquito control. The use of fungal biological pesticide, would trigger inevitable cross infection in silkworm, and lead to silkworm disease outbreak. Therefore, elucidation of the molecular mechanism of the interaction between entomopathogenic fungi and B. mori, and discovering novel antifungal molecules, would be beneficial to raising the antifungal ability of B. mori and has vast importance to agricultural pest control and cures for human fungal diseases.Entomopathogenic fungi penetrate the insect cuticle using its abundant hydrolases. These hydrolases, including cuticle-degrading protease and chitinase, both of them are important virulence factors. Overexpression of toxic protease can significantly enhance the virulence of pathogenic fungi. Although the basic model of fungal penetration of insect integument has been proposed, how do insects protect against fungal invasion and specific interactions between insects and fungi has yet to be further studied.The insect integument and hemolymph contain a large number of protease inhibitors, which are closely related to resistance against pathogenic microorganisms. To elucidate the molecular mechanism of insect resistance to fungal invasion and seek novel targets to improve silkworm antifungal ability, we focus on in-depth study of the serine protease inhibitors. In our previous study, microarray was used to screen expression-changed genes after microbial challenge. At least19serine protease inhibitors were up-regulated or down-regulated after microbial induced via oral. The preliminal results indicated that these serine protease inhibitors may be involved in resistance against pathogenic microorganisms. In particular, the expression level of various TIL-type protease inhibitors were significantly up-regulated after B. bassiana infection. Hence we can look for effective protein molecules against pathogenic fungus in order to enhance the antifungal ability of B. mori. In view of this, we cloned the full length cNDA of BmSPI38and BmSPI39from TIL family, expressed them in the prokaryotic cells, measured their activities and further studied the molecular mechanism of silkworm protease inhibitors against fungal invasion.3D structure prediction and site-directed mutagenesis were used to analyse the key sites for the inhibitory activity of BmSPI38and BmSPI39. Their physiological existence in the silkworm and the protection function of BmSPI39in the cocoon was researched systematically. The major results in this study are as follows:1. Full length cNDA clone, prokaryotic expression and activity assay of serine protease inhibitorsBased on the known EST sequences of BmSPI38and BmSPI39, we designed respectively four gene-specific primers for3’-and5’-RACE PCR. Finally, we obtained their fall length cDNA sequences. Both of BmSPI38and BmSPI39contain a single TIL domain with eight cysteines. Multiple sequence alignment showed that two cysteine(Cys2th and Cys6th) substitutions occurred in TIL domain, and the active sites are significantly different from protease inhibitors of other species. BmSPI38and BmSPI39were considered to be new members of the TIL family of SPIs due to its unique reactive sites and the numbers of conserved cysteines.Phylogenetic tree showed that all the TIL-type protease inhibitors were clustered into3branches, B. mori BmSPI35, BmSPI36, BmSPI37, BmSPI38, BmSPI39, BmSPI40and BmFPI-F (Fungal protease inhibitor-F) clustered into one clade (Group Ⅲ), Bombina bombina BbBSTI clustered into one clade (Group Ⅱ), other protease inhibitors clustered into one clade (Group Ⅰ). BmSPI38and BmSPI39have a farther evolutionary relationship to TIL-type protease inhibitors of other species, and shared high sequence identity with the BmFPI-F, suggesting that they might have similar functions to BmFPI-F in silkworm immunity.We obtained recombinant BmSPI38and BmSPI39with biological activity using prokaryotic expression technology. The inhibitory activity assay under different conditions of temperature or pH indicated that BmSPI38and BmSPI39have very high thermal and pH stability. To investigate the type of proteases that could be suppressed by recombinant BmSPI38and BmSPI39, various proteases were used in in vitro tests. The results showed that BmSPI38and BmSPI39can significantly suppress microbial proteases, such as subtilisin A and proteinase K, whereas it could not inhibit bovine pancreas trypsin, bovine pancreas chymotrypsin and elastase.2. Inhibitory activity of BmSPI38and BmSPI39against cuticle-degrading protease from fungi and dynamic characteristic analysisIn order to explore whether BmSPI38and BmSPI39are involved in protection from Entomopathogenic fungi, we choose the cuticle-degrading protease (CDEP-1) from B. bassiana for activity assays and in vitro binding experiments. The results showed that BmSPI38and BmSPI39can potently inhibit virulence protease CDEP-1of B. bassiana. RT-PCR analysis showed that BmSPI38was expressed highly in immune related tissues, such as head, integument and fatbody, suggesting that might suppress the integument penetration and invasion of silkworm by inhibiting cuticle-degrading proteases secreted by fungi. BmSPI39expressed specifically in the anterior and middle silk gland, may participate in some silk gland-related processes.Similar to Prl from M. anisopliae, CDEP-1induced the melanization of silkworm. Thus, CDEP-1-induced melanization can be blocked using BmSPI38or BmSPI39. This indicates that inhibitors could inhibit Prl-induced insecticidal melanization of silkworm and protect it. Precursor activation of phenoloxidase in vitro showed that CDEP-1cannot directly activate PPO, it may indirectly activate PPO by activating an upstream protein factor. Our results clarified another way to cope with fungal damage to silkworms, by blocking the harmful melanization caused by the protease produced by entomopathogenic fungi. A certain degree of melanization occurred in the hemolymph of silkworm108h after B. bassiana infection, and there was a postive correlation between blackening degree and concentration of conidia to some extent. Melanization associated with the phenoloxidase cascade, was involved in the immune response to fungal invasion.Subtilisin, protease K and cuticle-degrading protease CDEP-1used in this study belong to the Peptidases_S8family which contain a Peptidases_S8domain. Sequence analysis showed that members of Peptidases_S8family were highly similarity. The reported cuticle-degrading proteases are mainly belong to the PCSK9_ProteinaseK_like subfamily and Subtilisin_subset subfamily, are important fungal virulence factors. Based on the high similarity of subtilisin-like proteases and the intensive inhibitory activity of BmSPI38and BmSPI39against microbial proteases, these inhibitors might be useful for enhancing the anti-fungi ability of the silkworm.3. Protease inhibitors inhibit the invasion ofB. bassianaTo investigate whether BmSPI39is involved in the immune response to fungi, we use different concentrations of conidia suspension to infect silkworms, and performed a Western blot analysis of BmSPI39in the integument at different points after B. bassiana infection. The results showed that the protease inhibitor BmSPI39of silkworm was expressed in integument as tetramer. The expression of BmSPI39was up-regulated first, and then down-regulated, indicating that BmSPI39was involved in the immune response to fungi. Immunofluorescence experiments indicated that BmSPI39is mainly located in endocuticle and mesocuticle. In the light of the activity of fungal cuticle-degrading protease can be inhibited by BmSPI39, suggesting that protease inhibitors in integument can depress the penetration of fungi through inhibiting its cuticle-degrading proteases.In order to investigate whether protease inhibitors can enhance the antifungal ability of B. mori, recombinant BmSPI38and BmSPI39was used as an additive to spore suspension of B. bassiana, and treat silkworm by surface immersion. Each assay was done by application of B. bassiana alone or in conjunction with BSA, BmSPI38and BmSPI39. The experimental result indicated that the cumulative incidence of silkworm increased gradually with time after inoculation. By the fifth day, cumulative incidence rate of silkworm using B. bassiana alone was64.67%, and that using BSA, BmSPI38, BmSPI39as an additive was44.44%,18%and8%, respectively. We make observations on the silkworm at the5th day after infection. Most silkworm treated with B. bassiana alone or BSA as the additive displayed black damaged spots on the integument, darkened and matt, surface dehydration subsidence, inactivity and insensitivity to touch. The majority of silkworm treated with recombinant BmSPI38or BmSPI39as the additive have no obvious disease symptoms. Black spots emerged in the integument indicating that there is a cascade of phenoloxidase in silkworm, and the melanization reaction will be activated to participate in immune response against fungi, when infected by B. bassiana. Surprisingly, there were no obvious black spots on the intersegmental membrane of silkworm. This may be due to lack of the phenoloxidase system in intersegmental membrane, in which can not effeciently activate melanization. Through statistical analysis of survival rates of the five different treatment groups, BmSPI38and BmSPI39are able to significantly improve the survival rate of silkworm, suggesting that protease inhibitors could enhance the antifungal ability of silkworm, and can be used as a target resistant gene of silkworm.4. Analysis of key sites for inhibitory activity of BmSPI38and BmSPI39Multiple sequence alignment of TIL-type protease inhibitors from B. mori and other species showed that there are two cysteine substitutions in amino acid sequences of BmSPI38and BmSPI39. Therefore, we speculate that the amino acid residues in the two positions are the key sites to determine its inhibitory activity. Asp and Leu in the two positions were mutated to cysteines using site-directed mutagenesis technology. Structural models were constructed for three-dimensional structure of BmSPI39before and after mutation. The result shown that BmSPI39contains only2antiparallel (3sheets, as well as a small a helix, the rest are flexible loop regions. The whole structure contains four disulfide bonds (Cys29-Cys62, Cys40-Cys54, Cys44-Cys92, Cys64-Cys76), which maintain a rigid structure of protein stability and make the large flexible loop regions form a relatively compact and stable structure with certain flexibility. The superimposed structural models of BmSPI39and mutated BmSPI39Mu revealed that the two different mutagenic Cys are located in two adjacent loop regions, and they form an intramolecular disulfide bonds and make the two loop regions closer together. At the same time, Thr36m-O and Thr61m-N form a pair of hydrogen bonds to stabilize the structure changes. The disulfide bonds (Cys38m-Cys58m) and the adjacent new generating disulfide bonds bond (Cys40m-Cys54m) together to form a stable rigid region, and PI residue (Ala56m) is in the middle of the rigid region.The two amino acids in the BmSPI38and BmSPI39were mutated to cysteines using site-directed mutagenesis technology. The inhibitory activity of mutant BmSPI38Mu and BmSP139Mu against subtilisins was greatly reduced, indicating that cysteine substitutions in the two positions are an important cause of access to inhibitory ability against microbial protease. However. BmSPI38Mu and BmSPI39Mu didn’t obtain the ability to inhibit trypsin, chymotrypsin and elastase, suggesting that P1residues play an important role in determining its specificity. Base on the analysis of active sites and the number of cysteine in TIL-type protease inhibitors, we found that there is a great correlation of protease inhibitors specificity with the numbers of cysteine in TIL domain, properties and size of P1amino-acid residue. This study shows that replacement of P1residues by small molecules of neutral amino acids in silkworm TIL-type protease inhibitor, during the course of evolution, enable the inhibitors to gain a certain degree of inhibition against microbial protease, and cysteine substitutions enhance their inhibitory activity and specificity.5. Analysis of existing forms of the BmSPI38and BmSPI39in vivo and functions in cocoon shell protectionWe conducted an analysis of the recombinant BmSPI38and BmSPI39, using reducing and non-reducing SDS-PAGE electrophoresis, MALDI-TOF MS and activity staining. The results showed recombinant BmSPI38and BmSPI39were existing predominantly as polymers. Western blot analysis indicated that BmSPI38and BmSPI39expressed in various tissues and organs mainly in the form of tetramer, and they may execute function as polymers in silkworm.The RT-PCR and Western blot analysis revealed that BmSPI39is expressed specifically in the anterior and middle silk gland on the5th day of the fifth instar. It is supposed that BmSPI39was involved in some silk gland-related processes. BmSPI39was highly expressed in the silk gland prior to mounting, and secreted into the cocoon shell in the process of spinning silk. Our study showed that, protease inhibitor BmSPI39can inhibit microbial protease hydrolysis process of cocoon shell protein, hence provide a long-term effective protection for the prepupa and pupa.