Chemical proteomics reveals a second family of cyclic-di-AMP hydrolases.
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ABSTRACT:
SUBMITTER: Helmann JD
PROVIDER: S-EPMC4343114 | biostudies-literature | 2015 Feb
REPOSITORIES: biostudies-literature
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Chemical proteomics reveals a second family of cyclic-di-AMP hydrolases.
Proceedings of the National Academy of Sciences of the United States of America 20150130 7
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Project description:Streptococcus pneumoniae (the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In S. pneumoniae, c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K+ (Trk) family protein, CabP, which subsequently halts K+ uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δpde1 Δpde2 strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δpde1 Δpde2 strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δpde1 Δpde2 strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K+] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of cabP and trkH restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of S. pneumoniaeIMPORTANCE Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.
| S-EPMC6989799 | biostudies-literature
Project description:BackgroundConstant exposure of human gingival fibroblasts (HGFs) to oral pathogens trigger selective immune responses. Recently, the activation of immune response to cyclic dinucleotides (CDNs) via STING has come to the forefront. Reports show that other proteins outside the STING-TBK1-IRF3 axis respond to CDNs but a global view of impacted proteome in diverse cells is lacking. HGFs are constantly exposed to bacterial-derived cyclic-di-adenosine monophosphate (c-di-AMP) and cyclic-di-guanosine monophosphate (c-di-GMP).AimTo understand the response of HGFs to bacterial-derived CDNs, we carried out a global proteomics analysis of HGFs treated with c-di-AMP or c-di-GMP.MethodsThe expression levels of several proteins modulated by CDNs were examined.ResultsInterferon signaling proteins such as Ubiquitin-like protein ISG15 (ISG15), Interferon-induced GTP-binding protein Mx1 (MX1), Interferon-induced protein with tetratricopeptide repeats (IFIT) 1 (IFIT1), and (IFIT3) were significantly upregulated. Interestingly, other pathways not fully characterized to be regulated by CDNs, such as necroptosis signaling, iron homeostasis signaling, protein ubiquitination, EIF2 signaling, sumoylation and nucleotide excision repair pathways were also modulated by the bacterial-derived CDNs.ConclusionThis study has added to the increasing appreciation that beyond the regulation of cytokine production via STING, cyclic dinucleotides also broadly affect many critical processes in human cells.
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Project description:The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1β through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.
| S-EPMC3807221 | biostudies-literature
Project description:The Gram-positive model organism Bacillus subtilis produces the essential second messenger signaling nucleotide cyclic di-AMP. In B. subtilis and other bacteria, c-di-AMP has been implicated in diverse functions such as control of metabolism, cell division and cell wall synthesis, and potassium transport. To enhance our understanding of the multiple functions of this second messenger, we have studied the consequences of c-di-AMP accumulation at a global level by a transcriptome analysis. C-di-AMP accumulation affected the expression of about 700 genes, among them the two major operons required for biofilm formation. The expression of both operons was severely reduced both in the laboratory and a non-domesticated strain upon accumulation of c-di-AMP. In excellent agreement, the corresponding strain was unable to form complex colonies. In B. subtilis, the transcription factor SinR controls the expression of biofilm genes by binding to their promoter regions resulting in transcription repression. Inactivation of the sinR gene restored biofilm formation even at high intracellular c-di-AMP concentrations suggesting that the second messenger acts upstream of SinR in the signal transduction pathway. As c-di-AMP accumulation did not affect the intracellular levels of SinR, we conclude that the nucleotide affects the activity of SinR.
| S-EPMC4879592 | biostudies-literature
Project description:Cyclic diadenylate (c-di-AMP) is a widespread second messenger in bacteria and archaea that is involved in the maintenance of osmotic pressure, response to DNA damage, and control of central metabolism, biofilm formation, acid stress resistance, and other functions. The primary importance of c-di AMP stems from its essentiality for many bacteria under standard growth conditions and the ability of several eukaryotic proteins to sense its presence in the cell cytoplasm and trigger an immune response by the host cells. We review here the tertiary structures of the domains that regulate c-di-AMP synthesis and signaling, and the mechanisms of c-di-AMP binding, including the principal conformations of c-di-AMP, observed in various crystal structures. We discuss how these c-di-AMP molecules are bound to the protein and riboswitch receptors and what kinds of interactions account for the specific high-affinity binding of the c-di-AMP ligand. We describe seven kinds of non-covalent-π interactions between c-di-AMP and its receptor proteins, including π-π, C-H-π, cation-π, polar-π, hydrophobic-π, anion-π and the lone pair-π interactions. We also compare the mechanisms of c-di-AMP and c-di-GMP binding by the respective receptors that allow these two cyclic dinucleotides to control very different biological functions.
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Project description:Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.
| S-EPMC4521591 | biostudies-literature
Project description:Cyclic di-adenylate monophosphate (cyclic di-AMP) is an important second messenger in microorganisms. Cyclic di-AMP regulates bacterial cell volume and turgor via control of potassium and compatible solute transport but is also involved in many other processes, including the activation of the metazoan innate immune response to bacterial infections. We compare the activity of full-length membrane-embedded CdaA, the enzyme that synthesizes cyclic di-AMP, with the water-soluble catalytic domain CdaA-DAC. Purified CdaA from L. lactis was studied in the detergent-solubilized state, and in lipid nanodiscs and vesicles. We show that CdaA is tetrameric and the membrane-bound complex has more than 2-orders of magnitude higher activity than soluble CdaA-DAC. CdaA activity increases with pH but does not strongly depend on the salt or lipid content, factors that are crucial for the control of osmoregulatory transporters. Cryo-EM and in-silico structure prediction of CdaA show that the two DAC dimers engage in a head-to-head interaction, leading to cyclic-di-AMP formation. The inhibitor phosphoglucomutase prevents this active conformation. We observe dynamic flexibility between the catalytic and membrane domains, even in the presence of ATP or non-hydrolyzable substrate ApCpp. This is the first comprehensive functional and structural characterization of a full-length cyclic di-AMP-specific cyclase.
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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.
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Project description:Cyclic di-AMP (c-di-AMP) has been shown to play important roles as a second messenger in bacterial physiology and infections. However, understanding of how the signal is transduced is still limited. Previously, we have characterized a diadenylate cyclase and two c-di-AMP phosphodiesterases in Streptococcus pneumoniae, a Gram-positive pathogen. In this study, we identified a c-di-AMP binding protein (CabP) in S. pneumoniae using c-di-AMP affinity chromatography. We demonstrated that CabP specifically bound c-di-AMP and that this interaction could not be interrupted by competition with other nucleotides, including ATP, cAMP, AMP, phosphoadenylyl adenosine (pApA), and cyclic di-GMP (c-di-GMP). By using a bacterial two-hybrid system and genetic mutagenesis, we showed that CabP directly interacted with a potassium transporter (SPD_0076) and that both proteins were required for pneumococcal growth in media with low concentrations of potassium. Interestingly, the interaction between CabP and SPD_0076 and the efficiency of potassium uptake were impaired by elevated c-di-AMP in pneumococci. These results establish a direct c-di-AMP-mediated signaling pathway that regulates pneumococcal potassium uptake.
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Project description:Cyclic di-AMP (c-di-AMP) is an emerging second messenger in bacteria. It has been shown to play important roles in bacterial fitness and virulence. However, transduction of c-di-AMP signaling in bacteria and the role of c-di-AMP in biofilm formation are not well understood. The level of c-di-AMP is modulated by activity of di-adenylyl cyclase that produces c-di-AMP and phosphodiesterase (PDE) that degrades c-di-AMP. In this study, we determined that increased c-di-AMP levels by deletion of the pdeA gene coding for a PDE promoted biofilm formation in Streptococcus mutans. Deletion of pdeA upregulated expression of gtfB, the gene coding for a major glucan producing enzyme. Inactivation of gtfB blocked the increased biofilm by the pdeA mutant. Two c-di-AMP binding proteins including CabPA (SMU_1562) and CabPB (SMU_1708) were identified. Interestingly, only CabPA deficiency inhibited both the increased biofilm formation and the upregulated expression of GtfB observed in the pdeA mutant. In addition, CabPA but not CabPB interacted with VicR, a known transcriptional factor that regulates expression of gtfB, suggesting that a signaling link between CabPA and GtfB through VicR. Increased biofilm by the pdeA deficiency also enhanced bacterial colonization of Drosophila in vivo. Taken together, our studies reveal a new role of c-di-AMP in mediating biofilm formation through a CabPA/VicR/GtfB signaling network in S. mutans.
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