Expression And Functional Study Of Bombyx Mori (Silkworm) Ferritin-1

Lepidopteran insects provide important model systems for study of innate immunity of insects. Insect immune system consists of antimicrobial peptide production through IMD and Toll pathways, phenoloxidase (PO) cascade, and cellular responses. Silkworm (Bombyx mori) is the first lepidopteran insect which has been finished the genome sequence. Silkworm has been domesticated about 5000 years ago in China and does an important contribution to the economic growth of China.Iron is an essential element required for many cellular processes. Microbial infection affects iron distribution, which may serve as a key to understand the interaction between host and microbes. Ferritins and tranferrins, two major iron-binding proteins, are capable to maintain iron homeostasis of the host. Ferritins are iron-binding proteins with a hollow sphere structure, contains 24 subunits, and found in archeobacteria, eubacteria, plants, and invertebrates. Insect transferrins act as antioxidants and antimicrobial proteins to involve in innate immunity. BmFerl(Bombyx mori Ferritin 1) gene has three introns and one iron responsive element (IRE) in the 5’-untranslated region (UTR). Comparison of amino acid sequences of ferritin HCHs from fishes, crustaceans and insects shows that the seven residues required for ferroxidase activity are conserved. The phylogenetic tree of ferritin HCHs from various species clearly shows ferritin HCHs from insects are clustered into one group.To test whether BmFerl is involved in the silkworm defence against infection, two different bacteria, gram-positive strain Staphylococcus aureus and gram-negative Pseudomonas aeruginos, were injected into the hemocoel of larval silkworms. Then,-the iron concentration of silkworm hemolymph was determined, and quantitative real-time PCR (qRT-PCR) was used to analyze the transcriptional levels of BmFer-1 in the fat body and hemocytes. We found that bacterial infection strongly induced BmFerl transcription in hemocytes, but not in fat body. To investigate its physiological function, we expressed the mature BmFerl in E. coli. The recombinant protein formed inclusion body in the bacteria after induction by isopropyl-β-D-thiogalactoside (IPTG). The BmFerl protein was first isolated by nickel affinity chromatography under denaturing condition, and it was then purified and successfully refolded by serial dialysis against buffer with descending urea concentrations. SDS-PAGE and immunoblot were performed and results confirmed its purity and identity.Iron chelating assay showed that the purified recombinant BmFerl possesses iron-binding capacity with a concentration-dependant manner. Furthermore, the infection with bacteria (P. aeruginosa or S. aureus) also elevated the levels of iron in hemolymph and the BmFerl was found to be capable to lower down the iron level, suggesting that BmFerl functions as an iron chelator in the silkworm hemolymph. The iron is needed for the growth of bacteria and BmFerl limits it inside silkworm. The effect of iron on bacterial growth was also investigated, and it was found that addition of Fe2+ boosts bacterial growth when added to the culture medium. It was further revealed that sensitivities of P. aeruginosa and S. aureus to iron were different:P. aeruginosa was dependant on the iron concentration (0-64 μM) in the medium and higher level of FeCl2 does not stimulate the growth, and S. aureus is more sensitive to FeCl2 in the medium, but its growth was not affected by FeCl2 over 16 μM. After the bacteria-infected larvae with cured with the recombinant BmFerl, live bacteria counts in plasma were significantly lower 8 h later. It shows that bacteria use free iron to sustain their growth and BmFerl is able to suppress bacterial growth in plasma through limiting the iron access of the invading bacteria. We also found that BmFerl decreases the larval silkworm death which is caused by the infection of bacteria, suggesting that the BmFerl is helpful for larvae to combat with the pathogenic bacteria.