RNA interference of PHB1 enhances virulence of Vip3Aa to Sf9 cells and Spodoptera frugiperda larvae.

BACKGROUND: In our previous work, we demonstrated that prohibitin 2 (PHB2) on the membrane of Sf9 cells was a receptor for Vip3Aa, and PHB2 in mitochondria contributed to the mitochondrial stability to reduce Vip3Aa toxicity. Prohibitin 1 (PHB1), another prohibitin family member, forms heterodimers with PHB2 to maintain the structure and stability of mitochondria. To explore whether PHB1 impacts the action process of Vip3Aa, we examined the correlation between PHB1 and Vip3Aa virulence. RESULTS: We revealed that PHB1 did not colocalize with Vip3Aa in Sf9 cells. The pulldown and CoIP experiments confirmed that PHB1 interacted with neither Vip3Aa nor scavenger receptor-C (another Vip3Aa receptor). Downregulating phb1 expression in Sf9 cells did not affect the internalization of Vip3Aa but increased Vip3Aa toxicity. Further exploration revealed that the decrease of phb1 expression affected mitochondrial function, leading to increased ROS levels and mitochondrial membrane permeability and decreased mitochondrial membrane potential. The increase of mitochondrial cytochrome c release, caspase-3 activity and genomic DNA fragmentation implied that the apoptotic process was also affected. Finally, we applied RNAi to inhibit phb1 expression in Spodoptera frugiperda larvae. As a result, it significantly increased Vip3Aa virulence. CONCLUSION: We found that PHB1 was not a receptor for Vip3Aa but played an essential role in mitochondria. The downregulation of phb1 expression in Sf9 cells caused instability of mitochondria, and the cells were more prone to apoptosis when challenged with Vip3Aa. The combined use of Vip3Aa and phb1 RNAi showed a synergistic effect against S. frugiperda larvae. © 2023 Society of Chemical Industry.

Vegetative Insecticidal Protein Vip3Aa Is Transported via Membrane Vesicles in Bacillus thuringiensis BMB171.

Vegetative insecticidal protein Vip3Aa, secreted by many Bacillus thuringiensis (Bt) strains during the vegetative growth stage, represents the second-generation insecticidal toxin. In recent years, significant progress has been made on its structure and action mechanism. However, how it is translocated across the cytoplasmic membrane into the environment remains a mystery. This work demonstrates that Vip3Aa is not secreted by the General Secretion (Sec) System. To reveal the secretory pathway of Vip3A, we purified the membrane vesicles (MVs) of B. thuringiensis BMB171 and observed by TEM. The size of MVs was determined by the dynamic light scattering method, and their diameter was approximately 40-200 nm, which is consistent with the vesicles in Gram-negative bacteria. Moreover, Vip3A could be detected in the purified MVs by Western blot, and immunoelectron microscopy reveals Vip3A antibody-coated gold particles located in the MVs. After deleting its signal peptide, chitinase B (ChiB) failed to be secreted. However, the recombinant ChiB, whose signal peptide was substituted with the N-terminal 39 amino acids from Vip3A, was secreted successfully through MVs. Thus, this sequence is proposed as the signal region responsible for vesicle transport. Together, our results revealed for the first time that Vip3Aa is transported to the medium via MVs.

NupR Responding to Multiple Signals Is a Nucleoside Permease Regulator in Bacillus thuringiensis BMB171.

Nucleoside transport is essential for maintaining intracellular nucleoside and nucleobase homeostasis for living cells. Here, we identified an uncharacterized GntR/HutC family transcriptional regulator, NagR2, renamed NupR (nucleoside permease regulator), that mainly controls nucleoside transport in the Bacillus thuringiensis BMB171 strain. The deletion or overexpression of nupR affected the bacteria's utilization of guanosine, adenosine, uridine, and cytidine rather than thymidine. We further demonstrated that zinc ion is an effector for the NupR, dissociating NupR from its target DNA. Moreover, the expression of nupR is inhibited by NupR, ComK, and PurR, while it is promoted by CcpA. Also, a purine riboswitch located in its 5' noncoding region influences the expression of nupR. Guanine is the ligand of the riboswitch, reducing the expression of nupR by terminating the transcription of nupR in advance. Hence, our results reveal an exquisite regulation mechanism enabling NupR to respond to multiple signals, control genes involved in nucleoside transport, and contribute to nucleoside substance utilization. Overall, this study provides essential clues for future studies exploring the function of the NupR homolog in other bacteria, such as Bacillus cereus, Bacillus anthracis, Klebsiella pneumoniae, and Streptococcus pneumoniae. IMPORTANCE The transport of nucleosides and their homeostasis within the cell are essential for growth and proliferation. Here, we have identified a novel transcription factor, NupR, which, to our knowledge, is the first GntR family transcription factor primarily involved in the regulation of nucleoside transport. Moreover, responding to diverse intracellular signals, NupR regulates nucleoside transport. It is vital for utilizing extracellular nucleosides and maintaining intracellular nucleoside homeostasis. NupR may also be involved in other pathways such as pH homeostasis, molybdenum cofactor biosynthesis, nitrate metabolism, and transport. In addition, nucleosides have various applications, such as antiviral drugs. Thus, the elucidation of the transport mechanism of nucleosides could be helpful for the construction of engineered strains for nucleoside production.

PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability.

The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.

Attenuator LRR - a regulatory tool for modulating gene expression in Gram-positive bacteria.

With the rapid development of synthetic biology in recent years, particular attention has been paid to RNA devices, especially riboswitches, because of their significant and diverse regulatory roles in prokaryotic and eukaryotic cells. Due to the limited performance and context-dependence of riboswitches, only a few of them (such as theophylline, tetracycline and ciprofloxacin riboswitches) have been utilized as regulatory tools in biotechnology. In the present study, we demonstrated that a ribosome-dependent ribo-regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. Specifically, it offers a 60-fold change in expression in the presence of retapamulin and a low level of leaky expression of about 1-2% without antibiotics. Moreover, LRR can be combined with different promoters and performs well in Bacillus thuringiensis, B. cereus, B. amyloliquefaciens, and B. subtilis. Additionally, LRR also functions in the Gram-negative bacterium Escherichia coli. Furthermore, we demonstrate its ability to control melanin metabolism in B. thuringiensis BMB171. Our results show that LRR can be applied to regulate gene expression, construct genetic circuits and tune metabolic pathways, and has great potential for many applications in synthetic biology.

Autophagy induced by Vip3Aa has a pro-survival role in Spodoptera frugiperda Sf9 cells.

Vip3Aa is an insecticidal protein that can effectively control certain lepidopteran pests and has been used widely in biological control. However, the mechanism of action of Vip3Aa is unclear. In the present study, we showed that Vip3Aa could cause autophagy in Sf9 cells, which was confirmed by the increased numbers of GFP-Atg8 puncta, the appearance of autophagic vacuoles, and an elevated Atg8-II protein level. Moreover, we found that the AMPK-mTOR-ULK1 pathway is involved in Vip3Aa-induced autophagy, which might be associated with the destruction of ATP homeostasis in Vip3Aa-treated cells. Both the elevated p62 level and the increased numbers of GFP-RFP-Atg8 yellow fluorescent spots demonstrated that autophagy in Sf9 cells was inhibited at 24 h after Vip3Aa treatment. With the prolongation of Vip3Aa treatment time, this inhibition became more serious and led to autophagosome accumulation. Genetic knockdown of ATG5 or the use of the autophagy inhibitor 3-MA further increased the sensitivity of Sf9 cells to Vip3Aa. Overexpression of ATG5 reduced the cell mortality of Vip3Aa-treated cells. In summary, the results revealed that autophagy induced by Vip3Aa has a pro-survival role, which might be related to the development of insect resistance.

Mitochondria and Lysosomes Participate in Vip3Aa-Induced Spodoptera frugiperda Sf9 Cell Apoptosis.

Vip3Aa, a soluble protein produced by certain Bacillus thuringiensis strains, is capable of inducing apoptosis in Sf9 cells. However, the apoptosis mechanism triggered by Vip3Aa is unclear. In this study, we found that Vip3Aa induces mitochondrial dysfunction, as evidenced by signs of collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, release of cytochrome c, and caspase-9 and -3 activation. Meanwhile, our results indicated that Vip3Aa reduces the ability of lysosomes in Sf9 cells to retain acridine orange. Moreover, pretreatment with Z-Phe-Tyr-CHO (a cathepsin L inhibitor) or pepstatin (a cathepsin D inhibitor) increased Sf9 cell viability, reduced cytochrome c release, and decreased caspase-9 and -3 activity. In conclusion, our findings suggested that Vip3Aa promotes Sf9 cell apoptosis by mitochondrial dysfunction, and lysosomes also play a vital role in the action of Vip3Aa.