Aki Sagisaka

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.

Genome-wide analysis of host gene expression in the silkworm cells infected with Bombyx mori nucleopolyhedrovirus.

The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12h after BmNPV infection.

Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect anti-bacterial peptide

Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L-30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L-30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L-30R-NH2 fragment formed an alpha-helical structure in the lipidic environment. The anti-bacterial effect of the 19L-30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L-30R-NH2 fragment (8-10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L-30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A-30R-NH2) fragment and its analogues exhibited about 3-fold and 9-12-fold higher activity against S. aureus than did the 19L-30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

Correlation of Differential Expression of Silkworm Antimicrobial Peptide Genes with Different Amounts of Rel Family Proteins and Their Gene Transcriptional Activity

In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to κB sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.

DNA Vector-Based RNA Interference in Cell Lines Derived from Bombyx mori

Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

Expression profiling of novel bacteria-induced genes from the silkworm, Bombyx mori.

In this study, we have newly identified three bacteria-induced genes from the silkworm Bombyx mori by quantitative reverse transcriptase-polymerase chain reaction. One of these, eukaryotic initiation factor 4E-1 (eIF4E-1), is assumed to encode an eIF4E family, which plays a role in the initiation of translation as a mRNA cap-binding protein. The second gene is BmFOXG1, belonging to a family of forkhead transcription factors, FOXG1. The third gene is MBF2-related (MBF2-R) whose product has high homology to a co-activator protein MBF2 from B. mori. Although BmFOXG1 was up-regulated in the fat body in response to three kinds of bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, eIF4E-1 and MBF2-R were up-regulated by E. coli and B. subtilis, but not S. aureus, suggesting that bacteria possessing meso-diaminopimelic acid-containing peptidoglycan but not lysine-containing peptidoglycan activate eIF4E-1 and MBF2-R, probably through a conserved immune deficiency pathway. We further profiled the expression of three genes in different tissues and a silkworm cell line, NIAS-Bm-aff3, in response to bacteria, and at different times after bacterial challenge in the fat body.

Identification and functional analysis of pointed homologs in Bombyx mori

Using gene-knockdown techniques, we searched for endogenous Ets family proteins involved in the regulation of Escherichia coli-dependent lebocin promoter activation in the E. coli-responsive silkworm cell line NIAS-Bm-aff3. Results showed that the gene knockdown of BmPointeds (BmPNTs), Drosophila Pointed orthologs, enhanced E. coli-dependent lebocin promoter activation, suggesting that endogenous BmPNTs repress the activation of this promoter. Furthermore, we found that i) the BmPNT gene produced at least two alternative splicing isoforms, BmPNT1 and BmPNT2, both of which function as repressors; ii) BmPNTs were not associated with an already-reported repressor element, most proximal GGAA/T motif (EtsRE3), in lebocin promoter, which plays a role in the repression of E. coli- and BmRelish1-dependent lebocin promoter activation; iii) although BmPNTs did not directly affect BmRelish1-dependent lebocin promoter activation, they were able to directly repress its activation on the promoter lacking EtsRE3, probably because of competitive inhibition of binding of BmRelish1 to κB sites by BmPNTs; and iv) BmPNTs were mainly expressed in larval hemocytes, and the gene expression levels of BmPNT2, but not of BmPNT1, were decreased in response to E. coli and Bacillus subtilis. These findings suggest that endogenous BmPNTs are directly and indirectly involved in the repression of E. coli-mediated lebocin promoter activation in NIAS-Bm-aff3 cells.