Project description:Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many biological processes in bacteria. The genome in Mycobacterium tuberculosis encodes a single copy of the diguanylate cyclase gene (dgc) responsible for c-di-GMP synthesis. To determine the role of c-di-GMP signaling in M. tuberculosis, the mutant strain of Δdgc was generated in the virulent H37Rv strain. We used whole genome microarray expression profiling as a discovery platform to identify the genes controlled by c-di-GMP in M. tuberculosis, providing molecular proof for the phenotypes modulated by the signaling.
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.
Project description:Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many biological processes in bacteria. The genome in Mycobacterium tuberculosis encodes a single copy of the diguanylate cyclase gene (dgc) responsible for c-di-GMP synthesis. To determine the role of c-di-GMP signaling in M. tuberculosis, the mutant strain of Δdgc was generated in the virulent H37Rv strain. We used whole genome microarray expression profiling as a discovery platform to identify the genes controlled by c-di-GMP in M. tuberculosis, providing molecular proof for the phenotypes modulated by the signaling. Wild-type H37Rv and Δdgc cultures were analyzed under aerobic conditions or in an in vitro dormancy model. Bacteria were collected at OD600 =1.3 for the aerobic cultures and upon the beginning of anaerobiosis for the cultures in the dormancy model. One culture for each experiment was assayed except for Δdgc under anaerobiosis (2 independent cultures).
Project description:Transcriptomic analysis used to understand the influence of elevated intracellular cyclic-di-GMP in Shewanella oneidensis on expression of c-type cytochrome at transcript level.
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.
Project description:Sinorhizobium meliloti is a soil-dwelling symbiotic alphaproteobacterium. Cyclic di-GMP is an important second messenger controlling multiple functions in this microorganism. To understand transcriptional regulation by elevated c-di-GMP in S. meliloti, the transcriptome analysis was performed on the wild type strain S. meliloti Rm2011 carrying either an empty vector pWBT or diguanylate cyclase gene pleD overexpression plasmid pWBT-pleD.
Project description:<p>Cyclic di-GMP (c-di-GMP) is a well-known second messenger that plays a key role in many physiological processes in bacteria. The synthesis of lipids is essential for bacterial biofilm formation. However, whether c-di-GMP signaling modulates the synthesis of lipid and further regulates biofilm formation in mycobacteria is unclear, and the c-di-GMP receptor involved remains unknown. In this study, we characterized the nucleoid-associated protein (NAP) Lsr2 as a novel c-di-GMP receptor in mycobacteria. c-di-GMP specifically binds to Lsr2 at a ratio of 1:1. We showed that c-di-GMP promotes mycobacterial biofilm formation in a manner dependent on Lsr2. Furthermore, Lsr2 mediates the synthesis of keto-mycolic acid, the lipid component of the mycobacterial cell wall, by positively regulating the expression of HadD, a (3R)-hydroxyacyl-ACP dehydratase, thus, Lsr2 ultimately controls biofilm formation. Finally, c-di-GMP promotes the positive regulation of HadD by Lsr2 and mycobacterial biofilm formation. Thus, we report a novel c-di-GMP receptor that links the second messenger’s function to lipid synthesis and biofilm formation in mycobacteria.</p>
Project description:The cyanobacterial phytochrome Cph2 is a light-dependent diguanylate cyclase producing the second messenger c-di-GMP. Under blue light, the Cph2-dependent increase in the cellular c-di-GMP concentration leads to inhibition of motility in the cyanobacterium Synechocystis 6803. However, the targets of c-di-GMP in this cyanobacterium and its mechanism of action remained unclear. Here, we determined the cellular concentrations of three cyclic nucleotides in wild-type and Δcph2 cells after blue- and green light illumination. Inactivation of the photoreceptor gene completely abolished the blue-light dependent increase in the c-di-GMP content. Microarray analysis revealed that in the wild type in comparison to the Δcph2 mutant, blue light mainly led to a change in accumulation of mRNAs encoding minor pilins, putative chaperone usher pili as well as several chemotaxis regulators. The mRNA encoding the minor pilins pilA5-pilA6 is negatively affected by high c-di GMP content under blue light, whereas the minor pilin encoding operon pilA9-slr2018 accumulates under the same conditions, suggesting opposing functions of the respective gene sets. Based on mutational and gene expression analysis, we further suggest that the second Synechocystis 6803 homolog of a CRP-like transcription factor, SyCRP2, is the regulator of minor pilin gene expression and of putative chaperone usher pili genes slr1667/slr1668. Thus, our work indicates that the Cph2-mediated increase in cellular c-di-GMP concentration upon blue-light illumination specifically changes the transcriptome of Synechocystis 6803.
Project description:Pseudomonas aeruginosa is a Gram-negative bacterium able to survive and adapt in a multitude of niches as well as thriving within many different hosts. This versatility lies within a large genome of ca 6Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widely spread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. It is made by diguanylate cyclases and broken by phosphodiesterases while the intracellular level of this molecule drives phenotypic responses. The signaling involve modification of enzymes or proteins function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca 40 genes encoding putative DGC or PDE. The combined activity of those enzymes should reflect the c-di-GMP concentration while specific phenotypic output could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool, with essentially an impact on biofilm phenotypes including initial attachment and maturation, while very little is observed on motility which differs from previous studies. We observe that minor changes in c-di-GMP level have a drastic phenotypic impact thus supporting the idea of local vs global c-di-GMP pool. Our RNA-seq analysis indicates that all PAO1 dgc/pde genes are expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks owe to be reconstructed in one single strain and cannot be built by cross-comparison with studies in other strains.