Mamoru Hyodo

STING is a direct innate immune sensor of cyclic di-GMP

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1–IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS-GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk.

A comparative study between nanoparticle-targeted therapeutics and bioconjugates as obesity medication

Antiangiogenesis has been the focus of a new strategy for the treatment of obesity. However, little is known regarding the issue of whether targeting angiogenesis by nanoparticle-targeted therapeutic is advantageous or not in debugging the co-morbidity associated with diet-induced obesity (DIO) and the metabolic syndrome. We report herein on the positive effect of prohibitin (an adipose vascular marker)-targeted nanoparticle (PTNP) encapsulated in a proapoptotic peptide [(D)(KLAKLAK)₂, KLA] on DIO and dysfunctional adipose tissue, a major mediator of the metabolic syndrome, as evidenced by ectopic fat deposition. The systemic injection of DIO mice with a low dose of KLA-PTNP, rather than a bioconjugate composed of the same targeting peptide and KLA (Adipotide) resulted in a reduction in body weight, as evidenced by a significant decrease in serum leptin levels, in parallel with an antiobesity effect on dysfunctional adipose cells, including adipocytes and macrophages. In addition, the KLA-PTNP treatment resulted in a reduction in ectopic fat deposits in liver and muscle with the lipolytic action of elevated serum adiponectin, with no detectable hepatoxicity. Notably, drug delivery via PTNP that had accumulated in obese fat via the enhanced permeability and retention effect was enhanced by multivalent active targeting and cytoplasmic delivery into adipose endothelial cells via escaping from endosomes/lysosomes. Thus, vascular-targeted nanotherapy has the potential to contribute to the control of adipose function and ectopic fat deposition associated with obesity and the metabolic syndrome.

A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo.

Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing.

c-di-GMP (3′-5′-Cyclic Diguanylic Acid) Inhibits Staphylococcus aureus Cell-Cell Interactions and Biofilm Formation

ABSTRACT Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3′,5′-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillin-resistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms, while 5′-GMP had no major effect. We propose that cyclic dinucleotides such as c-di-GMP, used either alone or in combination with other antimicrobial agents, represent a novel and attractive approach in the development of intervention strategies for the prevention of biofilms and the control and treatment of infection.

Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3′,5′-Cyclic Diguanylic Acid

Cyclicdiguanylicacid(c-di-GMP;cGpGp)isaglobalsecondmessenger controlling motility and adhesion in bacterial cells. Intracellular concentrations of c-di-GMP depend on two opposite activities: diguanylate cyclase, recently assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase, associated with proteins harboring the EAL domain. To date, little is known about the targets of c-di-GMP in the cell or if it affects transcriptional regulation of certain genes. In order to expand our knowledge of the effect of this molecule on the bacterial metabolism, here we report on the Escherichia coli transcriptional profile under high levels of c-di-GMP. We show that an important number of genes encoding cell surface and membrane-bound proteins are altered in their transcriptional activity. On the other hand, genes encoding several transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of motility and cell division genes were altered, and consistent with this was the physiological analysis of cells overexpressing yddV,adiguanylatecyclase;thesecellsdisplayedanabnormalcelldivision process when high levels of c-di-GMP were present. We also show evidence that the diguanylate cyclase gene yddV is co-transcribed with dos,a heme base oxygen sensor with c-di-GMP-specific phosphodiesterase activity. A Δdos::kan mutation rendered the cells unable to divide properly,suggestingthatdosandyddVmaybepartofafine-tuningmechanism for regulating the intracellular levels of c-di-GMP.

3′,5′-Cyclic Diguanylic Acid Reduces the Virulence of Biofilm-Forming Staphylococcus aureus Strains in a Mouse Model of Mastitis Infection

ABSTRACT The cyclic dinucleotide 3′,5′-cyclic diguanylic acid (c-di-GMP) is a naturally occurring small molecule that regulates important signaling systems in bacteria. We have recently shown that c-di-GMP inhibits Staphylococcus aureus biofilm formation in vitro and its adherence to HeLa cells. We now report that c-di-GMP treatment has an antimicrobial and antipathogenic activity in vivo and reduces, in a dose-dependent manner, bacterial colonization by biofilm-forming S. aureus strains in a mouse model of mastitis infection. Intramammary injections of 5 and 50 nmol of c-di-GMP decreased colonization (bacterial CFU per gram of gland) by 0.79 (P > 0.05) and 1.44 (P < 0.01) logs, respectively, whereas 200-nmol doses allowed clearance of the bacteria below the detection limit with a reduction of more than 4 logs (P < 0.001) compared to the untreated control groups. These results indicate that cyclic dinucleotides potentially represent an attractive and novel drug platform which could be used alone or in combination with other agents or drugs in the prevention, treatment, or control of infection.

Ligand-based targeted delivery of a peptide modified nanocarrier to endothelial cells in adipose tissue

Ligand-based targeted delivery is an emerging platform in nanomedicine. We report herein on a peptide modified nanocarrier for a ligand-based targeted delivery system. The liposomal surface of the carrier was first modified with a linear peptide, followed by an adipose tissue-specific circular peptide (KGGRAKD) via a polyethylene glycol (PEG) spacer. To evaluate the specificity of the carrier, we purified primary cells from the endothelium of adipose tissue. The liposomes bound only to isolated primary cultured endothelial cells derived from inguinal adipose tissue (pcEC-IWAT) and not to other endothelial cell lines, such as MBEC-4 and MFLM-4. Cellular uptake was confirmed both qualitatively and quantitatively by confocal laser scanning microscopy (CLSM) and flow cytometry. The mechanism for the intracellular uptake of tPep-PEG-LPs into pcEC-IWAT, as evidenced by three independent experiments, involves saturation of receptor binding sites by excess free peptide, the blocking of receptors by an anti-prohibitin antibody and low temperature (4°C) experiments, resulting in the inhibition of uptake of tPep-PEG-LPs into pcEC-IWAT, suggesting that receptor mediated endocytosis largely contributed to the observed cellular uptake. A co-localization study using double labeled modified liposomes (lipid membrane: NBD-DOPE and aqueous phase: rhodamine) indicated that a predominant part of tPep-PEG-LPs was found without co-localization with lysosomes and retained their intactness. The selective delivery of tPep-PEG-LPs to endothelial cells in adipose tissue represents a potential approach for the design of diverse nanocarrier-based targeted delivery systems for targeting the vasculature in adipose tissue.

A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy

Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol=40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo.

Biochemical and physiological characterization of a BLUF protein-EAL protein complex involved in blue light-dependent degradation of cyclic diguanylate in the purple bacterium Rhodopseudomonas palustris.

Organisms adapt their physiologies in response to the quality and quantity of environmental light. Members of a recently identified photoreceptor protein family, BLUF domain proteins, use a flavin chromophore to sense blue light. Herein, we report that PapB, which contains a BLUF domain, controls the biofilm formation of the purple photosynthetic bacterium Rhodopseudomonas palustris. Purified PapB undergoes a typical BLUF-type photocycle, and light-excited PapB enhances the phosphodiesterase activity of the EAL domain protein, PapA, which degrades the second messenger, cyclic dimeric GMP (c-di-GMP). PapB directly interacts with PapA in vitro in a light-independent manner and induces a conformational change in the preformed PapA-PapB complex. A PapA-PapB docking simulation, as well as a site-directed mutagenesis study, identified amino acids partially responsible for the interaction between the PapA EAL domain and the two C-terminal α-helices of the PapB BLUF domain. Thus, the conformational change, which involves the C-terminal α-helices, transfers the flavin-sensed blue light signal to PapA. Deletion of papB in R. palustris enhances biofilm formation under high-intensity blue light conditions, indicating that PapB functions as a blue light sensor, which negatively regulates biofilm formation. These results demonstrate that R. palustris can control biofilm formation via a blue light-dependent modulation of its c-di-GMP level by the BLUF domain protein, PapB.