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: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:We have previously reported that Mycobacterium tuberculosis Rv2837c (cnpB) encodes a phosphodiesterase that specifically cleaves cyclic di-AMP (c-di-AMP) into AMP. Deletion of cnpB results in significant virulence attenuation in a mouse pulmonary infection model, which is very likely due to the significantly elevated c-di-AMP levels as overexpression of Mtb diadenylate cyclase, disA, also leads to a similar outcome. An earlier study also demonstrated that CnpB functions similarly to E. coli oligoribonuclease (Orn) that hydrolyzes 2-5-mer nanoRNAs (short oligonucleotides of five residues or shorter in length) except that CnpB prefers 2-mer nanoRNA as a substrate. Additionally, a recent report showed that CnpB also degrades cyclic di-GMP (c-di-GMP), although we demonstrated that CnpB prefers c-di-AMP to c-di-GMP according to an in vitro enzymatic kinetics analysis In this study, we initially attempted to determine c-di-AMP-mediated gene regulation in Mtb by comparing the expression profiles between WT and ∆cnpB using RNA-Seq. We found that the CRISPR-Cas system of M. tuberculosis was highly upregulated by deletion of cnpB.
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:c-di-GMP and c-di-AMP are important conserved second messenger in bacteria, they play a critical role in a wide range of cellular processes, such as motility, virulence, biofilm formation, cell-cycle progression and cell development, but there are only two c-di-GMP receptors and one c-di-AMP receptors were identified in mycobacteria so far, to identify more c-di-GMP and c-di-AMP receptors we compare the protein expression level of c-di-GMP or c-di-AMP synthesis enzyme overexpression strain with the wild type Mycobacterium smegmatis.
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: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:Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns (PAMPs) reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here we report that c-di-GMP comprehensively regulates both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs, while decreasing the number and repopulation capacity of long-term hematopoietic stem cells (LT-HSCs). Furthermore, we show that type I IFN acts as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while TGF-β1 is required for c-di-GMP-dependent splenic HSPC expansion. Our results define novel machinery underlying dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling.
Project description:Transcriptional profiling of gene expression between parental strain B31 and rrp1 mutant. Cyclic-di-GMP is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been widely recognized, the role of c-di-GMP in pathogen's life cycle in vector hosts is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for the production of c-di-GMP. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host, but could not survive in the tick vector. To identify the mechanisms of Rrp1 contributing to B. burgdorferi pathogenesis and gene regulation, microarray was employed to compare gene expression profiles between the parental strain B31 and the rrp1 mutant. Two-condition experiment, B31 vs. rrp1 mutant. Biological replicates: 3 B31, 3 rrp1 mutant, independently grown and harvested. One replicate (dye-swap) per array.