Shengkai Jin

Resistance to BmNPV via overexpression of an exogenous gene controlled by an inducible promoter and enhancer in transgenic silkworm, Bombyx mori.

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 105 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

A transgenic animal with antiviral properties that might inhibit multiple stages of infection

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms, causing severe economic losses in sericulture. To create antiviral silkworm strains, we constructed a transgenic vector in which the dsRNA for five tandem BmNPV genes was controlled by the BmNPV hr3 enhancer and IE1 promoter. The antivirus gene Bmlipase-1 was driven by B. mori midgut-specific promoter P2. Transgenic strains (SW-H) were generated via embryo microinjection using the practical silkworm strain SW. After infection with a high dose of BmNPV, the survival rates of SW-H and non-transgenic SW were 64% and 13%, respectively. SW-H could be the first transgenic animal that is highly antiviral and that might inhibit the virus at multiple stages of infection.

Identification of a midgut-specific promoter in the silkworm Bombyx mori

The midgut is an important organ for digestion and absorption of nutrients and immune defense in the silkworm Bombyx mori. In an attempt to create a tool for midgut research, we cloned the 1080 bp P2 promoter sequence (P2P) of a highly expressed midgut-specific gene in the silkworm. The transgenic line (P2) was generated via embryo microinjection, in which the expression of EGFP was driven by P2P. There was strong green fluorescence only in the midgut of P2. RT-PCR and Western blot showed that P2P was a midgut-specific promoter with activity throughout the larval stage. A transgenic truncation experiment suggested that regions -305 to -214 and +107 to +181 were very important for P2P activity. The results of this study revealed that we have identified a midgut-specific promoter with a high level of activity in the silkworm that will aid future research and application of silkworm genes.

Structural, evolutionary and functional analysis of APN genes in the Lepidoptera Bombyx mori.

Aminopeptidases N (APNs), the receptors of Bacillus thuringiensis (Bt) toxin in the lepidopteran midgut, are involved in the Bt pathogen infection mechanism. In the present work, we screened 102 APNs from SilkDB, ButterflyBase and MonarchBase; 16 APNs were identified from the silkworm (Bombyx mori) and 24 from the monarch butterfly (Danaus plexippus). Syntenic and phylogenetic tree analysis showed that APN genes have developed multi-family genes before evolutionary divergence of the Lepidoptera. The tissue-expression pattern shows some BmAPNs are specifically or highly expressed in the midgut. Bacillus bombysepticus (Bb) is a specific pathogen of B. mori, leading to acute fuliginosa septicemia of the larva. BmAPNs were modulated by real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis after Bb or Bt oral infection. There were different patterns of induced expression between Bb and Bt challenges, suggesting that B. mori has different responses to infection by the specific pathogen Bb and the nonspecific pathogen Bt. Research on BmAPNs will help us to better understand the evolutionary conservation and functions in Bb or Bt pathogen interaction with the host and to apply this knowledge in agricultural and forestry pest control.

Identification of a New Sprouty Protein Responsible for the Inhibition of the Bombyx mori Nucleopolyhedrovirus Reproduction

The rat sarcoma-extracellular signal regulated kinase mitogen-activated protein kinases pathway, one of the most ancient signaling pathways, is crucial for the defense against Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Sprouty (Spry) proteins can inhibit the activity of this pathway by receptor tyrosine kinases. We cloned and identified a new B. mori gene with a Spry domain similar to the Spry proteins of other organisms, such as fruitfly, mouse, human, chicken, Xenopus and zebrafish, and named it BmSpry. The gene expression analysis showed that BmSpry was transcribed in all of the examined tissues and in all developmental stages from embryo to adult. BmSpry also induced expression of BmNPV in the cells. Our results indicated: (1) the knock-down of BmSpry led to increased BmNPV replication and silkworm larvae mortality; (2) over-expression of BmSpry led to reduced BmNPV replication; and (3) BmSpry regulated the activation of ERK and inhibited BmNPV replication. These results showed that BmSpry plays a crucial role in the antiviral defense of the silkworm both in vitro and in vivo.

Complete Genome Sequence of Bacillus bombysepticus, a Pathogen Leading to Bombyx mori Black Chest Septicemia

ABSTRACT Bacillus bombysepticus is a Gram-positive spore-forming bacterium. Here, we announce the first complete genome sequence of this organism isolated from the cadavers of silkworm larvae that had been sick. The genome contains a single circular chromosome and a circular plasmid. Analyses of the B. bombysepticus genome will provide insights into its pathomechanisms and biology.

PC, a Novel Oral Insecticidal Toxin from Bacillus bombysepticus Involved in Host Lethality via APN and BtR-175

Insect pests have developed resistance to chemical insecticides, insecticidal toxins as bioinsecticides or genetic protection built into crops. Consequently, novel, orally active insecticidal toxins would be valuable biological alternatives for pest control. Here, we identified a novel insecticidal toxin, parasporal crystal toxin (PC), from Bacillus bombysepticus (Bb). PC shows oral pathogenic activity and lethality towards silkworms and Cry1Ac-resistant Helicoverpa armigera strains. In vitro assays, PC after activated by trypsin binds to BmAPN4 and BtR-175 by interacting with CR7 and CR12 fragments. Additionally, trypsin-activated PC demonstrates cytotoxicity against Sf9 cells expressing BmAPN4, revealing that BmAPN4 serves as a functional receptor that participates in Bb and PC pathogenicity. In vivo assay, knocking out BtR-175 increased the resistance of silkworms to PC. These data suggest that PC is the first protein with insecticidal activity identified in Bb that is capable of causing silkworm death via receptor interactions, representing an important advance in our understanding of the toxicity of Bb and the contributions of interactions between microbial pathogens and insects to disease pathology. Furthermore, the potency of PC as an insecticidal protein makes it a good candidate for inclusion in integrated agricultural pest management systems.

Genome-Wide Analysis of Host Responses to Four Different Types of Microorganisms in Bombyx Mori (Lepidoptera: Bombycidae)

Several pathogenic microorganisms have been used to investigate the genome-wide transcriptional responses of Bombyx mori to infection. However, studies have so far each focused on one microorganism, and systematic genome-wide comparison of transcriptional responses to different pathogenic microorganisms has not been undertaken. Here, we surveyed transcriptional responses of B. mori to its natural bacterial, viral, and fungal pathogens, Bacillus bombyseptieus, B. mori nucleopolyhedrovirus (BmNPV), and Beauveria bassiana, respectively, and to nonpathogenic Escherichia coli, by microarray analysis. In total, the expression of 2,436, 1,804, 1,743, and 912 B. mori genes was modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Notably, the expression of 620, 400, 177, or 165 of these genes was only modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, or E. coli, respectively. In contrast to the expression of genes related to juvenile hormone synthesis and metabolism, that of genes encoding juvenile hormone binding proteins was microorganism-specific. Three basal metabolic pathways were modulated by infection with any of the four microorganisms, and 3, 14, 5, and 2 metabolic pathways were specifically modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Interestingly, BmNPV infection modulated the JAK/STAT signaling pathway, whereas both the Imd and Toll signaling pathways were modulated by infection with B. bombyseptieus, B. bassiana, or E. coli. These results elucidate potential molecular mechanisms of the host response to different microorganisms, and provide a foundation for further work on host–pathogen interaction.

Genome-wide open chromatin regions and their effects on the regulation of silk protein genes in Bombyx mori

Nucleosome-depleted open chromatin regions (OCRs) often harbor transcription factor (TF) binding sites that are associated with active DNA regulatory elements. To investigate the regulation of silk-protein genes, DNA molecules isolated from the silk glands of third-day fifth-instar silkworm larvae and embryo-derived (BmE) cells were subjected to formal dehyde-assisted isolation of regulatory elements (FAIRE) and high-throughput sequencing. In total, 68,000 OCRs were identified, and a number of TF-binding motifs were predicted. In particular, OCRs located near silk-protein genes contained potential binding sites for functional TFs. Moreover, many TFs were found to bind to clusters of OCRs upstream of silk-protein genes, and to regulate the expression of these genes. The expression of silk protein genes may be related not only to regulating TFs (such as fkh, Bmdimm, and Bmsage), but also to developmental and hormone-induced TFs (such as zen, eve, Br, and eip74ef). Elucidation of genome-wide OCRs and their regulatory motifs in silk protein genes will provide valuable data and clues for characterizing the mechanisms of transcriptional control of silk protein genes.

Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.