Cyclic dimeric GMP(c-di-GMP)has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC,Gram-positive streptomycetes.Here,a diguanylate cyclase(DGC),CdgD,encoded by ...Cyclic dimeric GMP(c-di-GMP)has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC,Gram-positive streptomycetes.Here,a diguanylate cyclase(DGC),CdgD,encoded by SCO5345 from the model strain Streptomyces coelicolor,was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis.cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin,as well as completely blocked sporulation or aerial mycelium formation on two different solid media.In the cdgD-overexpression strain,intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain.In vitro enzymatic assay demonstrated that CdgD acts as a DGC,which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules.Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis.Collectively,our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.展开更多
The activation of the stimulating factor of the interferon gene(STING)pathway can enhance the immune response within the tumor.Cyclic diguanylate monophosphate(c-di-GMP)is a negatively charged,hydrophilic STING agonis...The activation of the stimulating factor of the interferon gene(STING)pathway can enhance the immune response within the tumor.Cyclic diguanylate monophosphate(c-di-GMP)is a negatively charged,hydrophilic STING agonist,however,its effectiveness is limited due to the poor membrane permeability and low bioavailability.Herein,we introduced KL-7 peptide derived from Aβamyloid fibrils that can self-assemble to form nanotubes to load and deliver c-di-GMP,which significantly enhanced c-di-GMP’s effectiveness and then exhibited a robust“in situ immunity”to kill melanoma cells.KL-7 peptide nanotube,also called PNT,was loaded with negatively charged c-di-GMP via electrostatic interaction,which prepared a nanocomposite named c-di-GMP-PNT.Treatment of RAW 264.7 cells(leukemia cells in mouse macrophage)with c-di-GMP-PNT markedly stimulated the secretion of IL-6 and INF-βalong with phospho-STING(Ser365)protein expression,indicating the activation of the STING pathway.In the unilateral flank B16-F10(murine melanoma cells)tumor-bearing mouse model,compared to PNT and cdi-GMP,c-di-GMP-PNT can promote the expression of INF-β,TNF-α,IL-6,and IL-1β.At the same time,up-regulated CD4 and CD8 active T cells kill tumors and enhance the immune response in tumor tissues,resulting in significant inhibition of tumor growth in tumor-bearing mice.More importantly,in a bilateral flank B16-F10 tumor model,both primary and distant tumor growth can also be significantly inhibited by c-di-GMP-PNT.Moreover,c-di-GMP-PNT demonstrated no obvious biological toxicity on the main organs(heart,liver,spleen,lung,and kidney)and biochemical indexes of mice.In summary,our study provides a strategy to overcome the barriers of free c-di-GMP in the tumor microenvironment and c-di-GMP-PNT may be an attractive nanomaterial for anti-tumor immunity.展开更多
C-di-GMP is one kind of second messengers which plays an important role not only in the regulation of various bacterial physiological activities but also in the activation of innate immune response to induce typeⅠint...C-di-GMP is one kind of second messengers which plays an important role not only in the regulation of various bacterial physiological activities but also in the activation of innate immune response to induce typeⅠinterferon in mammalian cells.In this assay,by using one-pot phosphoramidite method,three novel kinds of analogs of c-di-GMP including its phosphorthiates modified by ganciclovir(7 a,7 b,7 c)have been designed and synthesized.The immune-stimulatory results of these c-di-GMP analogs in RAW-Lucia ISG cells indicated that they couldn’t induce the release of typeⅠinterferon,which demonstrated that the intact structure of ribose moieties is very vital for their bioactivity upon the activation of STING signaling pathway.展开更多
基金supported by the National Natural Science Foundation of China (31630003, 31570072 and 31770088)the Science and Technology Commission of Shanghai Municipality (18ZR1446700)the National Science and Technology Major Project (2017ZX09101003-006-012)
文摘Cyclic dimeric GMP(c-di-GMP)has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC,Gram-positive streptomycetes.Here,a diguanylate cyclase(DGC),CdgD,encoded by SCO5345 from the model strain Streptomyces coelicolor,was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis.cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin,as well as completely blocked sporulation or aerial mycelium formation on two different solid media.In the cdgD-overexpression strain,intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain.In vitro enzymatic assay demonstrated that CdgD acts as a DGC,which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules.Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis.Collectively,our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.
基金supported by the National Natural Science Foundation of China(Nos.21877036 and 32201044)the Key Project of Developmental Biology and Breeding from Hunan Province(No.2022XKQ0205)+1 种基金the Hunan Natural Science Foundation(No.2021JJ40335)the Science and Technology Planning Project of Hunan Province(No.2018TP1017).
文摘The activation of the stimulating factor of the interferon gene(STING)pathway can enhance the immune response within the tumor.Cyclic diguanylate monophosphate(c-di-GMP)is a negatively charged,hydrophilic STING agonist,however,its effectiveness is limited due to the poor membrane permeability and low bioavailability.Herein,we introduced KL-7 peptide derived from Aβamyloid fibrils that can self-assemble to form nanotubes to load and deliver c-di-GMP,which significantly enhanced c-di-GMP’s effectiveness and then exhibited a robust“in situ immunity”to kill melanoma cells.KL-7 peptide nanotube,also called PNT,was loaded with negatively charged c-di-GMP via electrostatic interaction,which prepared a nanocomposite named c-di-GMP-PNT.Treatment of RAW 264.7 cells(leukemia cells in mouse macrophage)with c-di-GMP-PNT markedly stimulated the secretion of IL-6 and INF-βalong with phospho-STING(Ser365)protein expression,indicating the activation of the STING pathway.In the unilateral flank B16-F10(murine melanoma cells)tumor-bearing mouse model,compared to PNT and cdi-GMP,c-di-GMP-PNT can promote the expression of INF-β,TNF-α,IL-6,and IL-1β.At the same time,up-regulated CD4 and CD8 active T cells kill tumors and enhance the immune response in tumor tissues,resulting in significant inhibition of tumor growth in tumor-bearing mice.More importantly,in a bilateral flank B16-F10 tumor model,both primary and distant tumor growth can also be significantly inhibited by c-di-GMP-PNT.Moreover,c-di-GMP-PNT demonstrated no obvious biological toxicity on the main organs(heart,liver,spleen,lung,and kidney)and biochemical indexes of mice.In summary,our study provides a strategy to overcome the barriers of free c-di-GMP in the tumor microenvironment and c-di-GMP-PNT may be an attractive nanomaterial for anti-tumor immunity.
基金National Natural Science Foundation of China(Grant No.U1604285,21572013)
文摘C-di-GMP is one kind of second messengers which plays an important role not only in the regulation of various bacterial physiological activities but also in the activation of innate immune response to induce typeⅠinterferon in mammalian cells.In this assay,by using one-pot phosphoramidite method,three novel kinds of analogs of c-di-GMP including its phosphorthiates modified by ganciclovir(7 a,7 b,7 c)have been designed and synthesized.The immune-stimulatory results of these c-di-GMP analogs in RAW-Lucia ISG cells indicated that they couldn’t induce the release of typeⅠinterferon,which demonstrated that the intact structure of ribose moieties is very vital for their bioactivity upon the activation of STING signaling pathway.
