Isolation of ferritin and its interaction with BmNPV in the silkworm, Bombyx mori.

Ferritin is a ubiquitous iron storage protein that plays an important role in host defence against pathogen infections. In the present study, native ferritin was isolated from the hemolymph of Bombyx mori using native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that ferritin consisted of two subunits, designated as BmFerHCH and BmFerLCH. Previously integrated previous transcriptome and iTRAQ data showed that the two subunits were down-regulated in resistant silkworm strain BC9 and there was no obvious change in the expression levels of the subunits in susceptible silkworm strain P50 after BmNPV infection. Virus overlay assays revealed that B. mori ferritin as the form of heteropolymer had an interaction with B. mori nucleopolyhedrovirus (BmNPV), but it can't interact with BmNPV after depolymerisation. What's more, reverse transcription quantitative PCR (RT-qPCR) analysis suggested that BmFerHCH and BmFerLCH could be induced by bacteria, virus and iron. This is the first study to extract B. mori ferritin successfully and confirms their roles in the process of BmNPV infection. All these results will lay a foundation for further research the function of B. mori ferritin.

Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities.

A single-CRD C-type lectin is important for bacterial clearance in the silkworm.

C-type lectins (CTLs) depend on the carbohydrate-recognition domain (CRD) to recognize carbohydrates by a Ca(2+)-dependent mechanism. In animals, CTLs play critical roles in pathogen recognition, activation of the complement system and signaling pathways. Immulectins (Dual-CRD CTLs) in lepidopteran are involved in recognizing pathogens. However, little is known about the immune-related functions of insect single-CRD CTLs. Here, we reported the characterization of C-type lectin-S3 (CTL-S3), a single-CRD CTL from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S3 gene is 672 bp, which encodes a putative protein of 223 amino acids. CTL-S3 gene was expressed in a variety of tissues. Levels of CTL-S3 mRNA in fertilized eggs and whole larvae were elevated upon bacterial challenges. CTL-S3 was secreted to larval hemolymph. The recombinant protein (rCTL-S3) binds to bacterial cell wall components and bacteria. CTL-S3 inhibited the growth of Bacillus subtilis and caused agglutination of Staphylococcus aureus. More importantly, CTL-S3 facilitated the rapid clearance of Escherichia coli and Staphylococcus aureus from the body cavity of larvae. Taken together, our results suggested that CTL-S3 may function as an opsonin in larval hemolymph to enhance the clearance of pathogens.

Characterization of a dual-CRD galectin in the silkworm Bombyx mori.

Galectins (S-type lectins) are an ancient family of lectins with the ß-galactoside binding activity. In mammals, galectins play essential roles in many biological processes, such as development, immune homeostasis and tumor progression. However, few studies have been devoted to their functions in insects. Here, we characterized the only dual-CRD galectin in the silkworm Bombyx mori (BmGalectin-4). BmGalectin-4 cDNA possesses an open reading frame of 1089 bp, which encodes a putative galectin of 363 amino acids containing tandem carbohydrate recognition domains (CRDs). BmGalectin-4 was expressed in various tissues but the protein was most abundant in fertilized eggs. Its transcript level in fertilized eggs was upregulated upon bacterial challenge. Recombinant BmGalectin-4 purified from Escherichia coli bound to bacterial cell wall components and bacterial cells. In addition, the recombinant protein induced bacterial agglutination, but did not have antibacterial activity against selected microorganisms. Taken together, our results suggest that BmGalectin-4 may function as a pattern recognition receptor primarily in silkworm fertilized eggs.

Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori.

C-type lectins (CTLs) represent a large family of proteins that can bind carbohydrate moieties normally in a calcium-dependent manner. CTLs play important roles in mediating cell adhesion and the recognition of pathogens in the immune system. In the present study, we have identified 23 CTL genes in domestic silkworm Bombyx mori. CTL-domain proteins (CTLDPs) are classified into three groups based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures. These include twelve CTL-S (Single-CRD), six immulectins (Dual-CRD) and five CTL-X (CRD with other domains). We studied their phylogenetic features, analyzed the conserved residues, predicted tertiary structures, and examined the tissue expression profile and immune inducibility. Through bioinformatics analysis, we have putatively identified ten secretory and two cytoplasmic CTL-S; four secretory and two cytoplasmic immulectins; one secretory, one cytoplasmic and three transmembrane forms of CTL-X. Most B. mori CTLDPs form monophyletic groups with orthologs from Lepidoptera, Diptera, Coleoptera and Hymenoptera species. Immulectins of B. mori and Manduca sexta evolved from common ancestor genes perhaps due to gene duplication events of CTL-S ancestor genes. Homology modeling revealed that the overall structures of B. mori CTL domains are analogous to those of humans with a variable loop region. We examined the expression profile of CTLDP genes in naïve and immune-stimulated tissues. The expression and induction of CTLDP genes were related to the tissues and microorganisms. Together, our gene identification, sequence comparison, phylogenetic analysis, homology modeling and expression analysis laid a good foundation for the further studies of B. mori CTLDPs and comparative genomics.