Xiaojuan Deng

20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.

Molecular and functional characterization of a c-type lysozyme from the Asian corn borer, Ostrinia furnacalis

Abstract Some lepidopteran lysozymes have been reported to display activity against Gram-positive and Gram-negative bacteria, in contrast to most lysozymes that are active only against Gram-positive bacteria. OstrinLysC, a c-type lysozyme, was purified from the Asian corn borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae), and shows activity against Gram-positive and Gram-negative bacteria. The NH2-terminal amino acid sequence was determined by Edman degradation and used in a homology cloning strategy. The gene coding for OstrinLysC contains three exons and two introns. The expression profile of the OstrinlysC gene was examined by quantitative real-time PCR. Following injection of the larvae with bacteria, the OstrinlysC gene is strongly up-regulated in immune tissues. Transcripts were also detected in gut tissue. After feeding the larvae with bacteria, OstrinlysC transcripts increased in immune tissues. A very low level of transcript abundance was also detected in gut tissue. These results suggested that the OstrinlysC gene is involved in immune responses. The three dimensional structure of OstrinLysC was predicted. Based on comparison of the 3-D structure of OstrinLysC with that of silkworm lysozyme and chicken lysozyme, we hypothesize that the positive charge-rich surface and the short loop-2, which is close to the cluster of hydrophobic residues, may play important roles in the interaction with the outer membrane of Gram-negative bacterial cell walls.

BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

ABSTRACT Autophagy and apoptosis, which could be induced by common stimuli, play crucial roles in development and disease. The functional relationship between autophagy and apoptosis is complex, due to the dual effects of autophagy. In the Bombyx Bm-12 cells, 20-hydroxyecdysone (20E) treatment or starvation-induced cell death, with autophagy preceding apoptosis. In response to 20E or starvation, BmATG8 was rapidly cleaved and conjugated with PE to form BmATG8–PE; subsequently, BmATG5 and BmATG6 were cleaved into BmATG5-tN and BmATG6-C, respectively. Reduction of expression of BmAtg5 or BmAtg6 by RNAi decreased the proportion of cells undergoing both autophagy and apoptosis after 20E treatment or starvation. Overexpression of BmAtg5 or BmAtg6 induced autophagy but not apoptosis in the absence of the stimuli, but promoted both autophagy and apoptosis induced by 20E or starvation. Notably, overexpression of cleavage site-deleted BmAtg5 or BmAtg6 increased autophagy but not apoptosis induced by 20E or starvation, whereas overexpression of BmAtg5-tN and BmAtg6-C was able to directly trigger apoptosis or promote the induced apoptosis. In conclusion, being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in Bombyx Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis.

Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone

Resulted from alternative splicing of the 5′ exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5th larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5th larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

Gene Expression Divergence and Evolutionary Analysis of the Drosomycin Gene Family in Drosophila melanogaster

Drosomycin (Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes, Dro1, Dro2, Dro3, Dro4, Dro5, and Dro6, forming a multigene family on the 3L chromosome arm in Drosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the seven drosomycin genes, Drs, Dro2, Dro3, Dro4, and Dro5 showed constitutive expressions. Three out of five, Dro2, Dro3, and Dro5, were able to be upregulated by simple injury. Interestingly, Drs is an only gene strongly upregulated when Drosophila was infected with microbes. In contrast to these five genes, Dro1 and Dro6 were not transcribed at all in either noninfected or infected flies. Furthermore, by 5′ rapid amplification of cDNA ends, two transcription start sites were identified in Drs and Dro2, and one in Dro3, Dro4, and Dro5. In addition, NF-κB binding sites were found in promoter regions of Drs, Dro2, Dro3, and Dro5, indicating the importance of NF-κB binding sites for the inducibility of drosomycin genes. Based on the analyses of flanking sequences of each gene in D. melanogaster and phylogenetic relationship of drosomycins in D. melanogaster species-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates of drosomycin genes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.

Erratum to: Autophagy precedes apoptosis during the remodeling of silkworm larval midgut

Although several features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval–adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells.

Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

Abstract The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-IC) from Drosophila melanogaster and Thanatin from Podisus maculiventris , have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-IC , and Thanatin , were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-IC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris , which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-IC , and Thanatin , were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.

BmCalpains are involved in autophagy and apoptosis during metamorphosis and after starvation in Bombyx mori

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20‐hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain‐A/B with DmCalpain‐A/B, BmCalpain‐C with DmCalpain‐C, and BmCalpain‐7 with HsCalpain‐7. Then, the expression profiles of BmCalpains were analyzed by quantitative real‐time polymerase chain reaction, and results showed that expression of BmCalpain‐A/B, BmCalpain‐C and BmCalpain‐7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase‐1. Moreover, the apoptosis‐associated cleavage of BmATG6 in Bm‐12 cells was significantly enhanced when BmCalpain‐A/B and BmCalpain‐7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain‐A/B, ‐C and ‐7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis‐associated cleavage of BmATG6.

Regulation of antimicrobial peptide genes via insulin-like signaling pathway in the silkworm Bombyx mori

Antimicrobial peptides (AMPs) are important effector molecules of insect humoral immunity, and expression of AMPs is mainly regulated by the Toll and immune deficiency (IMD) pathways. FoxO, a key downstream regulator of the insulin-like signaling (ILS) pathway, has been recently reported to be involved in the regulation of AMPs in Drosophila melanogaster. In the present study, we investigated AMP gene expression and the regulation pathway controlled by the starvation in the silkworm Bombyx mori. We discovered that antibacterial activity in the hemolymph of B. mori larvae was increased by starvation, and expression of AMP genes (BmCecB6, BmAtta1, BmLeb3 and BmDefB) as well as the ILS target genes (FoxO, InR and Brummer) were strongly activated in the fat body by starvation. Moreover, phosphorylation of Akt kinase was reduced in the Bm-12 cells after starvation, suggesting that the ILS pathway was inhibited by starvation. We then showed that more FoxO protein was present in the cytoplasm than in the nucleus of Bm-12 cells under normal conditions, but more FoxO was detected in the nucleus after cells were starved for 8 h, indicating that FoxO was activated by starvation. In summary, our results indicated that starvation can activate AMP gene expression in B. mori via the ILS/FoxO signaling pathway.

An ML protein from the silkworm Bombyx mori may function as a key accessory protein for lipopolysaccharide signaling

ABSTRACT Lipopolysaccharide (LPS) is a common component of the outermost cell wall in Gram‐negative bacteria. In mammals, LPS serves as an endotoxin that can be recognized by a receptor complex of TLR4 (Toll‐like receptor 4) and MD‐2 (myeloid differentiation‐2) and subsequently induce a strong immune response to signal the release of tumor necrosis factor (TNF). In Drosophila melanogaster, no receptors for LPS have been identified, and LPS cannot activate immune responses. Here, we report a protein, BmEsr16, which contains an ML (MD‐2‐related lipid‐recognition) domain, may function as an LPS receptor in the silkworm Bombyx mori. We showed that antibacterial activity in the hemolymph of B. mori larvae was induced by Escherichia coli, peptidoglycan (PGN) and LPS and that the expression of antimicrobial peptide genes was also induced by LPS. Furthermore, both the expression of BmEsr16 mRNA in the fat body and the expression of BmEsr16 protein in the hemolymph were induced by LPS. Recombinant BmEsr16 bound to LPS and lipid A, as well as to PGN, lipoteichoic acid, but not to laminarin or mannan. More importantly, LPS‐induced immune responses in the hemolymph of B. mori larvae were blocked when the endogenous BmEsr16 protein was neutralized by polyclonal antibody specific to BmEsr16. Our results suggest that BmEsr16 may function as a key accessory protein for LPS signaling in B. mori. HIGHLIGHTSHighly purified LPS could induce the expression of antibacterial peptide in B. mori larvae at mRNA and protein level.BmEsr16 could bound to LPS and was induced expression by LPS in the fat body of B. mori larvae.Neutralization of BmEsr16 by antibody could block the LPS‐induced antibacterial activity in B. mori larvae.