Microbial Cyclic Di-Nucleotide Signaling
Microbial Cyclic Di-Nucleotide Signaling

Author: Shan-Ho Chou

Publisher: Springer Nature

Published: 2020-03-05

Total Pages: 640

ISBN-13: 3030333086

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This book explores the broad and diverse biological and physiological impacts of established and newly discovered cyclic di-nucleotide second messenger signaling systems, while also providing descriptions of the intriguing biochemical characteristics of multiple turnover enzymes and receptors. The respective chapters discuss the commonalities and diversity of cyclic di-GMP, cyclic di-AMP and recently discovered cyclic GMP-AMP signaling systems in manifold Gram-negative and Gram-positive bacteria. The global human pathogens Mycobacterium tuberculosis, Vibrio cholerae, Salmonella typhimurium, Escherichia coli and Streptococcus pneumoniae, the facultative human pathogen Pseudomonas aeruginosa, global plant pathogens as exemplified by Xanthomonas campestris and Burkholderia spp., and the omnipresent probiotic Lactobacilli, as well as environmentally important photoautotrophic cyanobacteria, the multicellular Myxococcus xanthus, and chemolithotrophic Acidithiobacillus are among the representatives of the microbial kingdom that are described. In turn, the various aspects of bacterial physiology affected by these signaling systems– e.g. biofilm formation and dispersal, the cell cycle, motility, virulence, production of antimicrobials, fundamental metabolism and osmohomeostasis – are discussed in detail in the context of different microorganisms. Dedicated chapters focus on the population diversity of cyclic dinucleotide signaling systems, their tendency to be horizontally transferred, the cyclic di-GMP signaling system in the social amoeba Dictyostelium, honorary cyclic (di)nucleotides, and the development of strategies for interfering with cyclic dinucleotide signaling in order to manipulate microbial behavior. Taken together, the chapters provide an authoritative source of information for a broad readership: beginners and advanced researchers from various disciplines; individuals seeking a broad overview of cyclic di-nucleotide signaling; and those who want to learn more about specific aspects. Also featuring reviews with a forward-looking perspective, the book offers a valuable source of inspiration for future research directions.

Microbial Cyclic Di-nucleotide Signaling
Microbial Cyclic Di-nucleotide Signaling

Author:

Publisher:

Published: 2020

Total Pages: 640

ISBN-13: 9783030333096

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This book explores the broad and diverse biological and physiological impacts of established and newly discovered cyclic di-nucleotide second messenger signaling systems, while also providing descriptions of the intriguing biochemical characteristics of multiple turnover enzymes and receptors. The respective chapters discuss the commonalities and diversity of cyclic di-GMP, cyclic di-AMP and recently discovered cyclic GMP-AMP signaling systems in manifold Gram-negative and Gram-positive bacteria. The global human pathogens Mycobacterium tuberculosis, Vibrio cholerae, Salmonella typhimurium, Escherichia coli and Streptococcus pneumoniae, the facultative human pathogen Pseudomonas aeruginosa, global plant pathogens as exemplified by Xanthomonas campestris and Burkholderia spp., and the omnipresent probiotic Lactobacilli, as well as environmentally important photoautotrophic cyanobacteria, the multicellular Myxococcus xanthus, and chemolithotrophic Acidithiobacillus are among the representatives of the microbial kingdom that are described. In turn, the various aspects of bacterial physiology affected by these signaling systems- e.g. biofilm formation and dispersal, the cell cycle, motility, virulence, production of antimicrobials, fundamental metabolism and osmohomeostasis - are discussed in detail in the context of different microorganisms. Dedicated chapters focus on the population diversity of cyclic dinucleotide signaling systems, their tendency to be horizontally transferred, the cyclic di-GMP signaling system in the social amoeba Dictyostelium, honorary cyclic (di)nucleotides, and the development of strategies for interfering with cyclic dinucleotide signaling in order to manipulate microbial behavior. Taken together, the chapters provide an authoritative source of information for a broad readership: beginners and advanced researchers from various disciplines; individuals seeking a broad overview of cyclic di-nucleotide signaling; and those who want to learn more about specific aspects. Also featuring reviews with a forward-looking perspective, the book offers a valuable source of inspiration for future research directions.

Development of RNA Sensors for Cyclic Dinucleotide Second Messengers in Bacteria
Development of RNA Sensors for Cyclic Dinucleotide Second Messengers in Bacteria

Author: Colleen Ann Kellenberger

Publisher:

Published: 2014

Total Pages: 125

ISBN-13:

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Second messengers are intracellular signaling molecules that enable cells to respond to environmental changes by relaying signals received at the cell surface to enact downstream physiological effects. A recently expanded class of second messengers in bacteria are the cyclic dinucleotides, which include cyclic di-GMP (cdiG), cyclic di-AMP (cdiA), and 3',3' cyclic AMP-GMP (cAG). In bacteria, these messengers regulate processes ranging from chemotaxis and sporulation to antibiotic resistance and virulence. Recent studies have found that bacterial cyclic dinucleotides, as well as a mammalian 2',3' cAG homolog, trigger the innate immune response upon detection in the mammalian cell cytosol. However, many components and roles of cyclic dinucleotide signaling remain unidentified or poorly understood. A clear understanding of cyclic dinucleotide signal transduction would further the pursuit of these signaling pathways as therapeutic targets and the effects of using cyclic dinucleotides as small molecule adjuvants. Herein, novel strategies for cyclic dinucleotide detection are reported that use that natural ability of riboswitch RNAs to discriminate between these nucleotide-based messengers. First, a microfluidic electrophoretic mobility shift assay (μMSA) is presented that enables highly sensitive and rapid detection of cdiG in vitro. This method takes advantage of the ligand-induced conformational change of a natural cdiG GEMM-I class riboswitch aptamer, termed Vc2. This same aptamer was then converted to a fluorescent biosensor for live-cell imaging by fusing it to the Spinach aptamer, an RNA mimic of GFP. This Vc2-Spinach RNA-based biosensor senses low levels of endogenous cdiG in Escherichia coli and responds to increased levels of cdiG upon coexpression of a heterologous cdiG synthase. A similar Spinach-based strategy was used to develop a cdiA RNA-based biosensor through fusion to riboswitches from the recently reported ydaO class. This cdiA sensor is the first tool for live cell imaging of cdiA and provided the first demonstration of the Spinach system in Listeria monocytogenes, an important gram-positive intracellular pathogen. Finally, as no known biosensor existed for 3',3' cAG, rational engineering of a cdiG aptamer led to the discovery of a subclass of GEMM-I riboswitches in Geobacter species that is naturally specific for 3',3' cAG. Further analysis revealed that this novel riboswitch subclass, which represents the first known effector for this messenger, is important in regulating the process of extracellular electron transfer in Geobacter and these riboswitches were used to develop the first biosensor specific for 3',3' cAG. It is envisioned that the use of these cyclic dinucleotide-specific sensors can be used to further identify unknown components in the signal transduction of these important regulatory molecules.

Synthesis of Non-Natural Cyclic Di-Nucleotides for the Investigation of Bacterial Signaling Pathways
Synthesis of Non-Natural Cyclic Di-Nucleotides for the Investigation of Bacterial Signaling Pathways

Author: Madison Hill Fletcher

Publisher:

Published: 2017

Total Pages: 273

ISBN-13:

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Humans navigate the world and interact with others through a complex series of communicative tools. We experience both internal and external stimuli, such as pangs of hunger or pain from an injury, and both verbal and nonverbal language. Bacteria also possess the ability to communicate, albeit in more discreet, yet no less complex ways. Bacteria rely on an incredibly diverse signaling system of triggers and responses in order to survive and to thrive. While we perceive language with our eyes and ears, bacteria employ a system of small molecules to relay both intra- and extracellular messages. They utilize this ability, known as quorum sensing to "talk" to their neighbors, express otherwise latent genetic characteristics, and to defend themselves against enemies. It has been suggested that this internal and external activity is linked, however, little is known about their interplay. This family of molecules, the cyclic di-nucleotides, which includes c-di-GMP and c-di-AMP, are critical to regulating bacterial processes such as motility, glucose remediation, and cell wall homeostasis. Their importance has spurred numerous investigations into their mechanism of action. Although found in very low concentrations within cells, they are capable of regulating a multitude of processes due to their ability to adopt variable conformations. To date, analog design by other groups has focused on the modification of the innate phosphate moiety as well as various substitutions or deletions at the 2'-position on the ribofuranose ring. However, these analogs have not been water soluble, limiting them to in vitro investigations only. We propose that by replacing the phosphate linkage entirely we can increase water solubility and have pursued a divergent total synthesis of various cyclic di-nucleotides featuring biomimetic linkages. Herein we address the methods we explored to optimize the synthesis of our three monomers, coupling strategies employed, the novel application of a Staudinger ligation to afford our abasic macrocycles and finally our progress towards implementing a bis-glycosylation strategy to install the desired nucleobase. We are able to efficiently provide large amounts of a di-amino, azide methyl ester, and N,O-substituted furanose monomers in no more than six steps from a common intermediate. These monomers are coupled and cyclized to form our four scaffolds, amide, carbamate, squaramide, and urea. Finally, we have begun to successfully implement our Brønsted acid mediated glycosylation strategy and understand its limitations. It is our goal to develop a general method to afford a diverse array of conformationally unique and water soluble cyclic di-nucleotide analogs with which to probe these essential bacterial signaling pathways.

Engineering New Methods to Investigate Cyclic Dinucleotides
Engineering New Methods to Investigate Cyclic Dinucleotides

Author: Alex J. Pollock

Publisher:

Published: 2021

Total Pages: 112

ISBN-13:

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Nucleotide second messengers are nucleotide-based small molecules which orchestrate cellular responses to internal cues and adaptation to environmental conditions. These messengers are ubiquitous throughout life and their flux coordinates diverse processes via binding response regulator proteins or riboswitches to induce post-translational, translational, or transcriptional responses. Some of the best studied nucleotide second messengers include cAMP which regulates carbon source utilization, (p)ppGpp which regulates biosynthetic metabolism in response to nutrient limitation, and 3′3′-c-di-GMP which regulates motility and biofilm production. Nucleotide second messengers are hypothesized to be ubiquitous because they are rapidly diffusible and can be quickly synthesized from and hydrolyzed into common substrates making them amenable to effective cellular coordination. This work focuses on the recently discovered nucleotide second messengers 3′3′-c-di-AMP and 2′3′-cGAMP which are respectively an essential bacterial molecule regulating virulence, cell wall homeostasis, and osmotic regulation and a mammalian innate immune signaling molecule signaling mis-localized double stranded DNA. These second messengers are synthesized by the condensation of two ATPs or an ATP and a GTP molecule, respectively, and have been the subject of increasing academic and clinical interest in the last decade. Despite significant advances in these fields, quantitative, single cell, and kinetic tools to detect these important nucleotide second messengers are lacking. This dearth has resulted in many important investigations being intractable. In response, we have generated FRET biosensors for both 2′3′-cGAMP and 3′3′-c-di-AMP. These adaptable biosensors allow for entirely new perspectives of bacterial and metazoan cyclic-di-nucleotide signaling. Additionally, we have also developed a luminescence-based coupled enzyme assay to facilitate higher-throughput population level quantification of 3′3′-c-di-AMP. We note that the recently discovered bacteriophage-defense signaling molecule, 3′3′-cGAMP can also be detected using this coupled enzyme assay but utility will be limited until a high affinity binding protein is found and adapted to extract 3′3′-cGAMP from complex samples. The tools developed in this work will facilitate diverse investigations of immune signaling and bacterial regulation with implications for immunotherapy, autoimmune disease, viral and bacterial restriction, and vaccine development.

GEMM-I Riboswitch-based Fluorescent Biosensors for Live Cell Analysis of Cyclic Dinucleotide Signaling
GEMM-I Riboswitch-based Fluorescent Biosensors for Live Cell Analysis of Cyclic Dinucleotide Signaling

Author: Xin Wang

Publisher:

Published: 2016

Total Pages: 124

ISBN-13:

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The bacterial second messenger cyclic di-GMP (c-di-GMP) holds a prominent position within the repertoire of bacterial signaling molecules. With its presence established in over 75% of sequence bacteria, c-di-GMP responds to primary environmental signals by affecting the bacterial lifestyle transition between the motile and sessile states, regulating biofilm formation, host colonization, and bacterial virulence. Over the past 30 years, many aspects of the c-di-GMP signaling pathway have become well characterized in no small part due to the plethora of tools that can quickly and conveniently detect c-di-GMP. Despite these triumphs, we have only begun to contend with the enormous scope of c-di-GMP signaling, an endeavor that would be greatly aided by new tools that are harder, better, faster, and stronger. The four fluorescent biosensors introduced herein bring us towards that goal. These second-generation RNA biosensors were designed based on a natural c-di-GMP riboswitch aptamer fused to the Spinach dye-binding aptamer, producing a fluorescent signal upon c-di-GMP binding. Their speed, sensitivity, and selectivity secure their place as a valuable tool for studying c-di-GMP signaling, with demonstrated efficacy in monitoring c-di-GMP in vivo in E. coli in a variety of conditions, including anaerobic and zinc-exposed environments. Furthermore, these sensors were adapted towards studying the related cyclic dinucleotide cyclic GMP-AMP (cGAMP), a recently discovered second messenger known for roles in bacterial intestinal colonization and surface sensing. Both c-di-G and cGAMP-specific biosensors were used to uncover components of the cGAMP signaling pathway in organisms not previously known to have cGAMP signaling. It is envisioned that these biosensors can be used to further understand c-di-GMP and cGAMP signaling in a variety of organisms, in vivo, in real time.

Cyclic Nucleotide Signaling
Cyclic Nucleotide Signaling

Author: Xiaodong Cheng

Publisher: CRC Press

Published: 2015-05-07

Total Pages: 282

ISBN-13: 1482235579

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Showcasing the recent progresses of the field, Cyclic Nucleotide Signaling covers the major tools and methodologies used in various areas of research. The majority of the chapters are protocol oriented, with the goal of providing clear directions for laboratory use. Students and investigators new to the field will find this book particularly inform

Bacterial Signaling
Bacterial Signaling

Author: Reinhard Krämer

Publisher: John Wiley & Sons

Published: 2009-12-09

Total Pages: 513

ISBN-13: 3527629246

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Providing a comprehensive insight into cellular signaling processes in bacteria with a special focus on biotechnological implications, this is the first book to cover intercellular as well as intracellular signaling and its relevance for biofilm formation, host pathogen interactions, symbiotic relationships, and photo- and chemotaxis. In addition, it deals in detail with principal bacterial signaling mechanisms -- making this a valuable resource for all advanced students in microbiology. Dr. Krämer is a world-renowned expert in intracellular signaling and its implications for biotechnology processes, while Dr. Jung is an expert on intercellular signaling and its relevance for biomedicine and agriculture.

c-di-GMP Signaling
c-di-GMP Signaling

Author: Karin Sauer

Publisher: Humana Press

Published: 2017-09-10

Total Pages: 486

ISBN-13: 9781493972395

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This volume provides a collection of protocols for the common experimental approaches used in the in the burgeoning field of c-di-GMP-dependent signaling. The chapters, divided into eight major parts, guide readers through methods on synthesis, detection, quantitation, modulation of the levels of c-di-GMP present in cells, procedures to detect and evaluate the interaction of c-di-GMP, and up and coming approaches focusing on the inhibition of c-di-GMP signaling.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, c-di-GMP Signaling: Methods and Protocols aims to inspire researchers to try new approaches.