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‣ Cyclic Di-GMP Stimulates Protective Innate Immunity in Bacterial Pneumonia▿

Karaolis, David K. R.; Newstead, Michael W.; Zeng, Xianying; Hyodo, Mamoru; Hayakawa, Yoshihiro; Bhan, Urvhashi; Liang, Hallie; Standiford, Theodore J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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Innate immunity is the primary mechanism by which extracellular bacterial pathogens are effectively cleared from the lung. We have previously shown that cyclic di-GMP (c-di-GMP [c-diguanylate]) is a novel small molecule immunomodulator and immunostimulatory agent that triggers protective host innate immune responses. Using a murine model of bacterial pneumonia, we show that local intranasal (i.n.) or systemic subcutaneous (s.c.) administration of c-di-GMP prior to intratracheal (i.t.) challenge with Klebsiella pneumoniae stimulates protective immunity against infection. Specifically, i.n. or s.c. administration of c-di-GMP 48 and 24 h prior to i.t. K. pneumoniae challenge resulted in significantly increased survival. Pretreatment with c-di-GMP resulted in a 5-fold reduction in bacterial CFU in the lung (P < 0.05) and an impressive >1,000-fold decrease in CFU in the blood (P < 0.01). c-di-GMP administration stimulated a robust innate response to bacterial challenge, characterized by enhanced accumulation of neutrophils and αβ T cells, as well as activated NK and αβ T lymphocytes, which was associated with earlier and more vigorous expression of chemokines and type I cytokines. Moreover, lung macrophages recovered from Klebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase and nitric oxide ex vivo than did macrophages isolated from infected mice pretreated with the control...

‣ Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA▿

Tamayo, Rita; Schild, Stefan; Pratt, Jason T.; Camilli, Andrew
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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In Vibrio cholerae, the second messenger cyclic di-GMP (c-di-GMP) positively regulates biofilm formation and negatively regulates virulence and is proposed to play an important role in the transition from persistence in the environment to survival in the host. Herein we describe a characterization of the infection-induced gene cdpA, which encodes both GGDEF and EAL domains, which are known to mediate diguanylate cyclase and c-di-GMP phosphodiesterase (PDE) activities, respectively. CdpA is shown to possess PDE activity, and this activity is regulated by its inactive degenerate GGDEF domain. CdpA inhibits biofilm formation but has no effect on colonization of the infant mouse small intestine. Consistent with these observations, cdpA is expressed during in vitro growth in a biofilm but is not expressed in vivo until the late stage of infection, after colonization has occurred. To test for a role of c-di-GMP in the early stages of infection, we artificially increased c-di-GMP and observed reduced colonization. This was attributed to a significant reduction in toxT transcription during infection. Cumulatively, these results support a model of the V. cholerae life cycle in which c-di-GMP must be down-regulated early after entering the small intestine and maintained at a low level to allow virulence gene expression...

‣ Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic-di-GMP in motility and virulence

Sultan, Syed Z.; Pitzer, Joshua E.; Miller, Michael R.; Motaleb, Md. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The genome of Borrelia burgdorferi encodes a set of genes putatively involved in cyclic-di-GMP metabolism. Although BB0419 was shown to be a diguanylate cyclase, the extent to which bb0419 or any of the putative cyclic-di-GMP metabolizing genes impact B. burgdorferi motility and pathogenesis has not yet been reported. Here we identify and characterize a phosphodiesterase (BB0363). BB0363 specifically hydrolyzed cyclic-di-GMP with a Km of 0.054 μM, confirming it is a functional cyclic-di-GMP phosphodiesterase. A targeted mutation in bb0363 was constructed using a newly developed promoterless antibiotic cassette that does not affect downstream gene expression. The mutant cells exhibited an altered swimming pattern, indicating a function for cyclic-di-GMP in regulating B. burgdorferi motility. Furthermore, the bb0363 mutant cells were not infectious in mice, demonstrating an important role for cyclic-di-GMP in B. burgdorferi infection. The mutant cells were able to survive within Ixodes scapularis ticks after a blood meal from naïve mice; however, ticks infected with the mutant cells were not able to infect naïve mice. Both motility and infection phenotypes were restored upon genetic complementation. These results reveal an important connection between cyclic-di-GMP...

‣ Cyclic di-GMP Signaling Regulates Invasion by Ehrlichia chaffeensis of Human Monocytes ▿ †

Kumagai, Yumi; Matsuo, Junji; Hayakawa, Yoshihiro; Rikihisa, Yasuko
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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Cyclic di-GMP (c-di-GMP) is a bacterial second messenger produced by GGDEF domain-containing proteins. The genome of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, encodes a single protein that contains a GGDEF domain, called PleD. In this study, we investigated the effects of c-di-GMP signaling on E. chaffeensis infection of the human monocytic cell line THP-1. Recombinant E. chaffeensis PleD showed diguanylate cyclase activity as it generated c-di-GMP in vitro. Because c-di-GMP is not cell permeable, the c-di-GMP hydrophobic analog 2′-O-di(tert-butyldimethylsilyl)-c-di-GMP (CDGA) was used to examine intracellular c-di-GMP signaling. CDGA activity was first tested with Salmonella enterica serovar Typhimurium. CDGA inhibited well-defined c-di-GMP-regulated phenomena, including cellulose synthesis, clumping, and upregulation of csgD and adrA mRNA, indicating that CDGA acts as an antagonist in c-di-GMP signaling. [32P]c-di-GMP bound several E. chaffeensis native proteins and two E. chaffeensis recombinant I-site proteins, and this binding was blocked by CDGA. Although pretreatment of E. chaffeensis with CDGA did not reduce bacterial binding to THP-1 cells, bacterial internalization was reduced. CDGA facilitated protease-dependent degradation of particular...

‣ Systematic analysis of cyclic di-GMP signaling enzymes and their role in biofilm formation and virulence in Yersinia pestis

Bobrov, Alexander G.; Kirillina, Olga; Ryjenkov, Dmitri A.; Waters, Christopher M.; Price, Paul A.; Fetherston, Jacqueline D.; Mack, Dietrich; Goldman, William E.; Gomelsky, Mark; Perry, Robert D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Cyclic di-GMP (c-di-GMP) is a signaling molecule that governs the transition between planktonic and biofilm states. Previously we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-β-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-β-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signaling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signaling network likely resulting from the different disease cycles of these human pathogens.

‣ Brucella melitensis Cyclic di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes▿†

Petersen, Erik; Chaudhuri, Pallab; Gourley, Chris; Harms, Jerome; Splitter, Gary
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Brucella melitensis encounters a variety of conditions and stimuli during its life cycle—including environmental growth, intracellular infection, and extracellular dissemination—which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.

‣ Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

Rybtke, Morten T.; Borlee, Bradley R.; Murakami, Keiji; Irie, Yasuhiko; Hentzer, Morten; Nielsen, Thomas E.; Givskov, Michael; Parsek, Matthew R.; Tolker-Nielsen, Tim
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2012 Português
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The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait...

‣ DDX41 recognizes bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response

Parvatiyar, Kislay; Zhang, Zhiqiang; Teles, Rosane M; Ouyang, Songying; Jiang, Yan; Iyer, Shankar S; Zaver, Shivam A; Schenk, Mirjam; Zeng, Shang; Zhong, Wenwan; Liu, Zhi-Jie; Modlin, Robert L; Liu, Yongjun; Cheng, Genhong
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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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 involving the STING adaptor, TBK1 kinase and IRF3 transcription factor. Here we identified the helicase DEAD box polypeptide 41 (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 shRNA in murine or human cells inhibited the induction of innate immune genes and resulted in defective STING, TBK1 and IRF3 activation in response to c-di-GMP or c-di-AMP. These results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by the DDX41 PRR, which complexes with STING to signal to TBK1-IRF3 and activate the interferon response.

‣ Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

Zorraquino, Violeta; García, Begoña; Latasa, Cristina; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione; Lasa, Iñigo; Solano, Cristina
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2013 Português
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Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation...

‣ 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...

‣ Occurrence of Cyclic di-GMP-Modulating Output Domains in Cyanobacteria: an Illuminating Perspective

Agostoni, Marco; Koestler, Benjamin J.; Waters, Christopher M.; Williams, Barry L.; Montgomery, Beronda L.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 13/08/2013 Português
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Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors—outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally...

‣ Cyclic di-GMP signalling and the regulation of bacterial virulence

Ryan, Robert P.
Fonte: Society for General Microbiology Publicador: Society for General Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2013 Português
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Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3′-5′)-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small ‘adaptor’ protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein–protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence.

‣ Exploring Environmental Control of Cyclic di-GMP Signaling in Vibrio cholerae by Using the Ex Vivo Lysate Cyclic di-GMP Assay (TELCA)

Koestler, Benjamin J.; Waters, Christopher 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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Vibrio cholerae senses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. V. cholerae encodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system in V. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named the ex vivo lysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show that V. cholerae grown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP...

‣ Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production

Srivastava, Disha; Hsieh, Meng-Lun; Khataokar, Atul; Neiditch, Matthew B.; Waters, Christopher M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homolog of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.

‣ ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa

Jones, Christopher J.; Newsom, David; Kelly, Benjamin; Irie, Yasuhiko; Jennings, Laura K.; Xu, Binjie; Limoli, Dominique H.; Harrison, Joe J.; Parsek, Matthew R.; White, Peter; Wozniak, Daniel J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 06/03/2014 Português
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The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon...

‣ Mechanism of activation of bacterial cellulose synthase by cyclic-di-GMP

Morgan, Jacob L. W.; McNamara, Joshua T.; Zimmer, Jochen
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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The bacterial signaling molecule cyclic-di-GMP stimulates the synthesis of bacterial cellulose, frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of the cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the cyclic-di-GMP-activated BcsA–B complex. The structures reveal that cyclic-di-GMP releases an auto-inhibited state of the enzyme by breaking a salt bridge which otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Additionally, the cyclic-di-GMP activated BcsA–B complex contains a nascent cellulose polymer whose terminal glucose unit rests at a novel location above BcsA’s active site where it is positioned for catalysis. Our mechanistic insights are the first examples of how cyclic-di-GMP allosterically modulates enzymatic functions.

‣ Identification of a cyclic-di-GMP-modulating response regulator that impacts biofilm formation in a model sulfate reducing bacterium

Rajeev, Lara; Luning, Eric G.; Altenburg, Sara; Zane, Grant M.; Baidoo, Edward E. K.; Catena, Michela; Keasling, Jay D.; Wall, Judy D.; Fields, Matthew W.; Mukhopadhyay, Aindrila
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 29/07/2014 Português
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We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn2+-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However...

‣ Novel Cyclic di-GMP Effectors of the YajQ Protein Family Control Bacterial Virulence

An, Shi-qi; Caly, Delphine L.; McCarthy, Yvonne; Murdoch, Sarah L.; Ward, Joseph; Febrer, Melanie; Dow, J. Maxwell; Ryan, Robert P.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/10/2014 Português
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Bis-(3′,5′) cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (Kd∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes...

‣ Stimulation of Innate Immunity by In Vivo Cyclic di-GMP Synthesis Using Adenovirus

Koestler, Benjamin J.; Seregin, Sergey S.; Rastall, David P. W.; Aldhamen, Yasser A.; Godbehere, Sarah; Amalfitano, Andrea; Waters, Christopher M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2014 Português
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The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

‣ Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

Roelofs, Kevin G.; Jones, Christopher J.; Helman, Sarah R.; Shang, Xiaoran; Orr, Mona W.; Goodson, Jonathan R.; Galperin, Michael Y.; Yildiz, Fitnat H.; Lee, Vincent T.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 27/10/2015 Português
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Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele...