Takashi Iwasaki

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.

Selective cancer cell cytotoxicity of enantiomeric 9-mer peptides derived from beetle defensins depends on negatively charged phosphatidylserine on the cell surface

Four enantiomeric 9-mer peptides named d-peptide A, B, C and D were designed and synthesized on the basis of 43-mer insect defensins from two beetles. The d-9-mer peptides maintained bacterial membrane disruptive activity similar to the original peptides and also showed various extents of growth inhibitory activity against different cancer cell lines. Of these peptides, d-peptide B exhibited the highest selective cancer cell cytotoxicity against the mouse myeloma cell line, P3-X63-Ag8.653. Flow cytometric and scanning electron microscopic analysis revealed d-peptide B disrupts mouse myeloma membrane construction, whereas no cytotoxic effect on normal leukocytes was observed. Moreover, a strong correlation between negatively charged phosphatidylserine (PS) density in cancer cell membrane surface and sensitivity to d-9-mer peptides were observed in various cancer cell lines. These results suggest that d-9-mer peptides have negative charge-dependent selective cancer cell cytotoxicity targeting PS in the cancer cell membrane. In addition, synergic growth inhibitory activity against mouse myeloma was observed in combinations of d-peptide B and dexamethasone. These results suggest d-9-mer peptides are promising candidates for novel anticancer drugs.

Gene Expression of a Novel Defensin Antimicrobial Peptide in the Silkworm, Bombyx mori

cDNA encoding a novel defensin (BmDefensinB) was cloned from the fat body of the silkworm, Bombyx mori, and gene expression was analyzed. BmDefensinB showed typical structural characteristics of invertebrate defensins. Phylogenetic and bootstrap analyses indicated that it has no orthologs, whereas previously reported BmDefensinA is the ortholog of Spodoptera frugiperda (Sf)Spodoptericin. The BmDefensinB gene was expressed tissue-specifically in the fat body and was strongly activated by bacteria such as Escherichia coli and Bacillus subtilis, and by an entomopathogenic fungus Beauveria bassiana. In contrast, the BmDefensinA gene was expressed to a much lesser extent. Expression of the BmDefensinB gene was strongly stimulated by B. mori Rel proteins RelB and Relish, supporting the observation that this gene is activated by E. coli, B. subtilis, and B. bassiana. These results suggest that BmDefensinB gene expression is controlled through both the Toll and the Imd pathway, and that this gene plays an important role in B. mori immune reactions against infection by bacteria and fungi.

Physiological function, expression pattern, and transcriptional regulation of a Caenorhabditis elegans insulin-like peptide, INS-18.

In Caenorhabditis elegans, insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an important pathway that controls larval diapause and adult lifespan. The IIS pathway is modulated by many insulin-like peptides (ILPs) through the DAF-2 receptor, the sole insulin/IGF-1 receptor-like protein in C. elegans. We previously identified the ILP, INS-18, and predicted its tertiary structure to be similar to the crystal structures of human insulin and IGF-1. In this study, the physiological function of INS-18 was first examined by gene disruption and overexpression, and we identified INS-18 as a DAF-2 antagonist required for larval diapause and longevity. Analysis of the INS-18 expression pattern using a reporter gene showed it to be expressed in nerve cells, including hermaphrodite-specific neurons (HSNs) at the adult stage. Other ILP expressions have not been previously observed in HSNs, and we believe that INS-18 expression in these cells may contribute to longevity by regulating reproduction. Loss of the DAF-16 transcription factor located downstream of the IIS pathway completely blocked ins-18 expression. We propose a positive feedback model for the regulation of ins-18 expression in which an antagonist binding to the DAF-2 receptor increases ins-18 gene expression, thus leading to increased INS-18 protein levels and increased DAF-2 receptor binding. Thus, this study provides a new insight into the hormonal regulation of insulin, an important and widespread process in the animal kingdom.

Development of a bioactive fiber with immobilized synthetic peptides designed from the active site of a beetle defensin.

The 9-mer peptides RLYLRIGRR and RLLLRIGRR were immobilized to amino-functionalized cotton fibers by a modification of the SPOT synthesis technique. The antibacterial activities of the peptide-immobilized cotton fibers against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) were investigated. Antibacterial assays revealed that these fibers inhibit the growth of MRSA and the antibacterial activities were maintained after washing and sterilization by autoclaving. The anticancer effect of the peptide-immobilized fiber was also investigated with mouse myeloma cells and human leukemia cells. These results indicate that these fibers have strong growth inhibition activity against bacteria and cancer cells.

Cellular uptake and in vivo distribution of polyhistidine peptides.

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

A Caenorhabditis elegans Insulin-Like Peptide, INS-17: Its Physiological Function and Expression Pattern

The insulin/insulin-like growth factor-1 signaling pathway of Caenorhabditis elegans regulates larval diapause and adult lifespan through the sole insulin receptor-like protein, DAF-2. In the present study, the physiological function and expression pattern of INS-17, one of the C. elegans insulin-like peptides, were examined by disruption and overexpression of the gene, and by the use of a reporter gene. INS-17 might function as a DAF-2 antagonist in the regulation of larval diapause, but not of the adult lifespan. The reporter protein was intensively expressed during larval diapause. It showed a drastic decrease in amount after larval diapause, which matches well the physiological function of INS-17.

Characteristics of Novel Insect Defensin-Based Membrane-Disrupting Trypanocidal Peptides

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH2), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.

An altered method of feeding RNAi that knocks down multiple genes simultaneously in the nematode Caenorhabditis elegans.

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.

Diapause is associated with a change in the polarity of secretion of insulin-like peptides

The insulin/IGF-1 signalling (IIS) pathway plays an important role in the regulation of larval diapause, the long-lived growth arrest state called dauer arrest, in Caenorhabditis elegans. In this nematode, 40 insulin-like peptides (ILPs) have been identified as putative ligands of the IIS pathway; however, it remains unknown how ILPs modulate larval diapause. Here we show that the secretory polarity of INS-35 and INS-7, which suppress larval diapause, is changed in the intestinal epithelial cells at larval diapause. These ILPs are secreted from the intestine into the body cavity during larval stages. In contrast, they are secreted into the intestinal lumen and degraded during dauer arrest, only to be secreted into the body cavity again when the worms return to developmental growth. The process that determines the secretory polarity of INS-35 and INS-7, thus, has an important role in the modulation of larval diapause.