Virulent and necrotrophic strategies of Bacillus thuringiensis in susceptible and resistant insects, Galleria mellonella.

Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at ∼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48 h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Antibacterial Mechanism of Gloverin2 from Silkworm, Bombyx mori.

Gloverin is one of the glycine-rich antimicrobial peptide exclusively found in Lepidoptera insects. It is generally activated through the innate immune system in insects. In this study, recombinant Gloverin2 from Bombyx mori (BmGlv2) was synthesized using a prokaryotic expression system. Circular dichroism spectroscopy showed that the recombinant BmGlv2 has random coil structure, which is relatively stable at the temperatures ranging from 15 to 82.5 °C. Antimicrobial activity analysis revealed that BmGlv2 significantly inhibited the growth of gram-negative bacteria, Escherichia coli JM109 and Pseudomonas putida, by disrupting cell integrity. Western blotting and immunofluorescence analyses suggested that BmGlv2 absorbed on the cell surface after incubation, which might be the first step in the antibacterial process. Our results also proved that the cell wall component lipopolysaccharides (LPS) induce a conformational change in BmGlv2 from a random coil to α-helix. Subsequently, α-helical BmGlv2 would recruit more BmGlv2 and form higher aggregation state. Collectively, these findings expand our understanding of antibacterial mechanism of BmGlv2.

Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression.

The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without using fetal bovine serum and/or feeder cells pretreated by irradiation or exposure to mitomycin. Highly productive clones of Drosophila melanogaster S2 cells were prepared in a two-step procedure, comprising the establishment of a polyclonal population and subsequent single cell isolation by limiting dilution. Necessary growth factors were provided by co-cultivation of single transformants with untransfected feeder cells, which were later removed by antibiotic selection. Enhanced expression of EGFP and two target peptides was confirmed by flow cytometry and dot/western blotting. Highly productive clones were stable, showed a uniform expression profile and typically a sixfold to tenfold increase in cell-specific productivity.

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.

Antimicrobial proteins and peptides (AMPs) are valuable as leads in the pharmaceutical industry for the development of novel anti-infective drugs. Here we describe the efficient heterologous expression and basic characterization of a Gloverin-family AMP derived from the greater wax moth Galleria mellonella. Highly productive single-cell clones prepared by limiting dilution achieved a 100% increase in productivity compared to the original polyclonal Drosophila melanogaster S2 cell line. Comprehensive screening for suitable expression conditions using statistical experimental designs revealed that optimal induction was achieved using 600 µM CuSO4 at the mid-exponential growth phase. Under these conditions, 25 mg/L of the AMP was expressed at the 1-L bioreactor scale, with optimal induction and harvest times ensured by dielectric spectroscopy and the online measurement of optical density. Gloverin was purified from the supernatant by immobilized metal ion affinity chromatography followed by dialysis. In growth assays, the purified protein showed specific antimicrobial activity against two different strains of Escherichia coli.

Molecular cloning and characterization of gloverin from the diamondback moth, Plutella xylostella L. and its interaction with bacterial membrane.

Gloverin restricted to Lepidoptera is known to be a glycine-rich and heat stable antimicrobial protein. The current research reports a 650 bp full-length cDNA encoding gloverin from Plutella xylostella (PxGlo) by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. PxGlo transcript was detected in both developmental stages and several tissues of 4th instar naïve larvae of P. xylostella with higher levels in the fat bodies. The mRNA levels of PxGlo increased appreciably in fat bodies after injection of Escherichia coli K12. The recombinant PxGlo expressed in S2 cells was purified by Anti-V5 M2 agarose beads which showed high activity against E. coli K12, while low activity against Bacillus thuringiensis, Staphylococcus aureus and E. coli D31. The analysis of transmission electron microscope and scan electron microscope showed PxGlo to cause significant morphological alteration in the E. coli K12 cell surface. Knockdown of PxGlo expression by RNAi increased the larval susceptibility towards the pathogenic bacteria i.e., Serratia marcescens and B. thuringiensis. Our results showed that PxGlo is an inducible antibacterial peptide which exhibits high activity mainly against E. coli K12, and PxGlo performs vital roles against the infection of pathogenic bacteria.

Conserved microRNA miR-8 blocks activation of the Toll pathway by upregulating Serpin 27 transcripts.

microRNAs (miRNAs) play significant regulatory roles in gene expression at the post-transcriptional level. This includes modulating processes such as development, immunity, cancer, and host-pathogen interactions. It was recently shown that the phylogenetically deeply conserved miRNA, miR-8, plays a role in maintaining the homeostasis of immunity by suppressing the production of anti-microbial peptides. In this study, we show that miR-8 from the insect Plutella xylostella positively regulates the transcript levels of the serine protease inhibitor Serpin 27, which has been shown to regulate activation of the Toll pathway and prophenoloxidase involved in the melanization response in insects. Interestingly, miR-8 is downregulated following parasitization by Diadegma semiclausum leading to significant declines in Serpin 27 transcript levels. This allows upregulation of antimicrobial peptides, such as gloverin, that are controlled by the Toll pathway and activation of proteolytic cascades essential for humoral immune responses to foreign invasion.

Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury

Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80 percent identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.

Expression of antibacterial peptide gloverin in vitro / 中国临床药理学与治疗学

AIM: To construct a recombinant vector containing gloverin and express it in vitro by Rapid Translation System(RTS500). METHODS: The gloverin cDNA was amplified by PCR and inserted into the prokaryotic expression vector pIVEX2.3. The recombinant product was identified by PCR and enzyme digestion. The positive reconstructed expression plasmid pIVEX2.3-G was expressed by RTS500 in vitro. The expressed protein was identified by SDS-PAGE and western blotting. RESULTS: Positive recombinant plasmid pIVEX2.3-G was successfully constructed. Target protein of 13.8 Kda with 6-his tag was detected by SDS-PAGE and western blotting. CONCLUSION: Gloverin polypeptide is successfully expressed in inactive E.coli in vitro.

Prokaryotic expression, purification and identification of anti-bacterial peptide gloverin / 第三军医大学学报

Objective To express anti-bacterial peptide gloverin in prokaryotic system and investigate its bioactivity. Methods E.coli BL21 was used to express gloverin. Western blot was used to identify the production and Tachypleus Amebocyte Lysate (TAL) was used to detect the bioactivity of the production. Results The production was identified as gloverin peptide by Western blotting, and the TAL data indicated that the production could neutralize LPS. Conclusion Anti-bacterial peptide gloverin is successfully expressed in E.coli BL21.