Bacterial second messengers, cGMP and c-di-GMP, in a quest for regulatory dominance.
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ABSTRACT:
SUBMITTER: Gomelsky M
PROVIDER: S-EPMC3770956 | biostudies-literature | 2013 Sep
REPOSITORIES: biostudies-literature
Publications
Bacterial second messengers, cGMP and c-di-GMP, in a quest for regulatory dominance.
Gomelsky Mark M Galperin Michael Y MY
The EMBO journal 20130820 18
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Project description:Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.
| S-EPMC3775879 | biostudies-literature
Project description:Cyclic-di-GMP and cyclic-di-AMP are second messengers produced by bacteria and influence bacterial cell survival, differentiation, colonization, biofilm formation, virulence, and bacteria-host interactions. In this study, we show that in both RAW264.7 macrophage cells and primary bone marrow-derived macrophages, the production of IFN-β and IL-6, but not TNF, in response to cyclic-di-AMP and cyclic-di-GMP requires MPYS (also known as STING, MITA, and TMEM173). Furthermore, expression of MPYS was required for IFN response factor 3 but not NF-κB activation in response to these bacterial metabolites. We also confirm that MPYS is required for type I IFN production by cultured macrophages infected with the intracellular pathogens Listeria monocytogenes and Francisella tularensis. However, during systemic infection with either pathogen, MPYS deficiency did not impact bacterial burdens in infected spleens. Serum IFN-β and IL-6 concentrations in the infected control and MPYS(-/-) mice were also similar at 24 h postinfection, suggesting that these pathogens stimulate MPYS-independent cytokine production during in vivo infection. Our findings indicate that bifurcating MPYS-dependent and -independent pathways mediate sensing of cytosolic bacterial infections.
| S-EPMC3159690 | biostudies-literature
Project description:The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.
| S-EPMC3501571 | biostudies-literature
Project description:Twenty-five years have passed since the discovery of cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.
| S-EPMC3591986 | biostudies-literature
Project description:The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) regulates cellular motility and the synthesis of organelles and molecules that promote adhesion to a variety of biological and nonbiological surfaces. These properties likely require tight spatial and temporal regulation of c-di-GMP concentration. We have developed genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors to monitor c-di-GMP concentrations within single bacterial cells by microscopy. Fluctuations of c-di-GMP were visualized in diverse Gram-negative bacterial species and observed to be cell cycle dependent. Asymmetrical distribution of c-di-GMP in the progeny correlated with the time of cell division and polarization for Caulobacter crescentus and Pseudomonas aeruginosa. Thus, asymmetrical distribution of c-di-GMP was observed as part of cell division, which may indicate an important regulatory step in extracellular organelle biosynthesis or function.
| S-EPMC3906730 | biostudies-literature
Project description:UnlabelledThe alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Δrel(Msm) and ΔdcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015,http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Δrel(Msm) and ΔdcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria.ImportanceOur work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p)ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Δrel(Msm) and ΔdcpA strains are defective in biofilm formation. In this work, the overproduction of (p)ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p)ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p)ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.
| S-EPMC4836230 | biostudies-literature
Project description:As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.
| S-EPMC6745502 | biostudies-literature
Project description:The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.
| S-EPMC4591828 | biostudies-literature
Project description:Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.
| S-EPMC5116723 | biostudies-literature
Project description:The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics.The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. Here we provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by cyclic-di- GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, NF-!B and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways.Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand. Three-condition experiment: macrophages transfected with mono-GMP (negative control), double-stranded DNA (positive control), or cyclic-di-GMP (experimental condition). Biological replicates: two, independently treated, harvested, and hybridized to arrays. One replicate per array, except two technical replicates were performed for one of the positive control samples.
2009-07-21 | E-GEOD-16943 | biostudies-arrayexpress