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‣ Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence

Kulesekara, Hemantha; Lee, Vincent; Brencic, Anja; Liberati, Nicole; Urbach, Jonathan; Miyata, Sachiko; Lee, Daniel G.; Neely, Alice N.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Ausubel, Frederick M.; Lory, Stephen
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes...

‣ Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR

De, Nabanita; Pirruccello, Michelle; Krasteva, Petya Violinova; Bae, Narae; Raghavan, Rahul Veera; Sondermann, Holger
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains...

‣ The Anaplasma phagocytophilum PleC Histidine Kinase and PleD Diguanylate Cyclase Two-Component System and Role of Cyclic Di-GMP in Host Cell Infection▿

Lai, Tzung-Huei; Kumagai, Yumi; Hyodo, Mamoru; Hayakawa, Yoshihiro; Rikihisa, Yasuko
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[32P]GMP detected an ∼47-kDa endogenous protein...

‣ Effect of cyclic bis(3′–5′)diguanylic acid and its analogs on bacterial biofilm formation

Ishihara, Yuka; Hyodo, Mamoru; Hayakawa, Yoshihiro; Kamegaya, Taichi; Yamada, Keiko; Okamoto, Akira; Hasegawa, Tadao; Ohta, Michio
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Publicado em /12/2009 Português
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Cyclic bis(3′–5′)diguanylic acid (cyclic-di-GMP) functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes. We measured cyclic-di-GMP and its structural analogs such as cyclic bis(3′–5′)guanylic/adenylic acid (cyclic-GpAp), cyclic bis(3′–5′)guanylic/inosinic acid (cyclic-GpIp) and monophosphorothioic acid of cyclic-di-GMP (cyclic-GpGps) for effects on the biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa. We constructed a knockout mutant of SA0701, which is a GGDEF motif protein relevant to diguanylate cyclase from S. aureus 2507. We confirmed that the biofilm formation of this mutant (MS2507ΔSA0701) was reduced. Cyclic-di-GMP corresponding to physiological intracellular levels given in the culture recovered the biofilm formation of MS2507ΔSA0701, whereas its analogs did not, indicating that unlike a previous suggestion, cyclic-di-GMP was involved in the positive regulation of the biofilm formation of S. aureus and its action was structurally specific. At a high concentration (200 μM), cyclic-di-GMP and its analogs showed suppression effects on the biofilm formation of S. aureus and P. aeruginosa, and according to the quantification study using costat analysis...

‣ The Atypical Two-component Sensor Kinase Lpl0330 from Legionella pneumophila Controls the Bifunctional Diguanylate Cyclase-Phosphodiesterase Lpl0329 to Modulate Bis-(3′-5′)-cyclic Dimeric GMP Synthesis

Levet-Paulo, Mélanie; Lazzaroni, Jean-Claude; Gilbert, Christophe; Atlan, Danièle; Doublet, Patricia; Vianney, Anne
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotranfer to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether...

‣ Type IV Pilus Assembly in Pseudomonas aeruginosa over a Broad Range of Cyclic di-GMP Concentrations

Jain, Ruchi; Behrens, Anna-Janina; Kaever, Volkhard; Kazmierczak, Barbara I.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2012 Português
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Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103ΔfimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the ΔfimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103ΔfimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle...

‣ Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy

Stelitano, Valentina; Brandt, Annegret; Fernicola, Silvia; Franceschini, Stefano; Giardina, Giorgio; Pica, Andrea; Rinaldo, Serena; Sica, Filomena; Cutruzzolà, Francesca
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3′–5′ cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3′–5′ cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.

‣ Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora

Edmunds, Adam C.; Castiblanco, Luisa F.; Sundin, George W.; Waters, Christopher M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
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The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition...

‣ Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

Huangyutitham, Varisa; Güvener, Zehra Tüzün; Harwood, Caroline S.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 07/05/2013 Português
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WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated.

‣ PdeB, a Cyclic Di-GMP-Specific Phosphodiesterase That Regulates Shewanella oneidensis MR-1 Motility and Biofilm Formation

Chao, Lily; Rakshe, Shauna; Leff, Maija; Spormann, Alfred M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2013 Português
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Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore...

‣ A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis

An, Shi-Qi; Chin, Ko-Hsin; Febrer, Melanie; McCarthy, Yvonne; Yang, Jauo-Guey; Liu, Chung-Liang; Swarbreck, David; Rogers, Jane; Maxwell Dow, J; Chou, Shan-Ho; Ryan, Robert P
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo-complex, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di-GMP signalling.

‣ Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia; Vianney, Anne
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2014 Português
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Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins...

‣ Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence

Chaudhuri, Swarnava; Pratap, Siddharth; Paromov, Victor; Li, Zhijun; Mantri, Chinmay K.; Xie, Hua
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2014 Português
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Porphyromonas gingivalis is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3′-5′) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in P. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the pgn_1932 gene had a significant effect on the intracellular c-di-GMP level in P. gingivalis. Genetic analysis showed that expression of the fimA and rgpA genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the pgn_1932 mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the pgn_1932 gene also had a significant impact on the adhesive and invasive capabilities of P. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by P. gingivalis by controlling the expression and biosynthesis of FimA.

‣ The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Moscoso, Joana A.; Jaeger, Tina; Valentini, Martina; Hui, Kailyn; Jenal, Urs; Filloux, Alain
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2014 Português
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Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes...

‣ High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases

Lieberman, Ori J.; Orr, Mona W.; Wang, Yan; Lee, Vincent T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide...

‣ Cyclic-di-GMP signalling regulates motility and biofilm formation in Bordetella bronchiseptica

Sisti, Federico; Ha, Dae-Gon; O'Toole, George A.; Hozbor, Daniela; Fernández, Julieta
Fonte: Society for General Microbiology Publicador: Society for General Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
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The signalling molecule bis-(3′–5′)-cyclic-dimeric guanosine monophosphate (c-di-GMP) is a central regulator of diverse cellular functions, including motility, biofilm formation, cell cycle progression and virulence, in bacteria. Multiple diguanylate cyclase and phosphodiesterase-domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) modulate the levels of the second messenger c-di-GMP to transmit signals and obtain such specific cellular responses. In the genus Bordetella this c-di-GMP network is poorly studied. In this work, we evaluated the expression of two phenotypes in Bordetella bronchiseptica regulated by c-di-GMP, biofilm formation and motility, under the influence of ectopic expression of Pseudomonas aeruginosa proteins with EAL or GGDEF domains that regulates the c-di-GMP level. In agreement with previous reports for other bacteria, we observed that B. bronchiseptica is able to form biofilm and reduce its motility only when GGDEF domain protein is expressed. Moreover we identify a GGDEF domain protein (BB3576) with diguanylate cyclase activity that participates in motility and biofilm regulation in B. bronchiseptica. These results demonstrate for the first time, to our knowledge, the presence of c-di-GMP regulatory signalling in B. bronchiseptica.

‣ A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP ▿

Holland, Linda M.; O'Donnell, Sinéad T.; Ryjenkov, Dmitri A.; Gomelsky, Larissa; Slater, Shawn R.; Fey, Paul D.; Gomelsky, Mark; O'Gara, James P.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore...

‣ BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14▿ †

Kuchma, Sherry L.; Brothers, Kimberly M.; Merritt, Judith H.; Liberati, Nicole T.; Ausubel, Frederick M.; O'Toole, George A.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene...

‣ Deletion Mutant Library for Investigation of Functional Outputs of Cyclic Diguanylate Metabolism in Pseudomonas aeruginosa PA14

Ha, Dae-Gon; Richman, Megan E.; O'Toole, George A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2014 Português
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We constructed a library of in-frame deletion mutants targeting each gene in Pseudomonas aeruginosa PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

‣ Diguanylate Cyclases Control Magnesium-Dependent Motility of Vibrio fischeri▿

O'Shea, Therese M.; Klein, Adam H.; Geszvain, Kati; Wolfe, Alan J.; Visick, Karen L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
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Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB, respectively, for magnesium-dependent induction of flagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we introduced the wild-type genes on high-copy plasmids into Escherichia coli. We assayed for the production of cyclic di-GMP using two-dimensional thin-layer chromatography and found that strains carrying these plasmids produced a small but reproducible spot that migrated with an Rf value consistent with cyclic di-GMP that was not produced by strains carrying the vector control. Disruptions of mifA or mifB increased flagellin levels...