Search

Cyclic diguanylate (c-di-GMP) is a near-ubiquitous signaling molecule that regulates the motility-to-sessility transition in many bacterial species. Among the phenotypes influenced by c-di-GMP are biofilm formation, motility, cell cycle, and virulence. The hallmark phenotypes regulated by c-di-GMP—biofilm formation and motility—are key determinants of host–bacterial interactions. A large body of research has identified the roles of c-di-GMP in regulating phenotypes in culture. While numerous studies have investigated roles for c-di-GMP during the establishment and maintenance of pathogenic host–bacterial associations, considerably less attention has been devoted to defining the roles of c-di-GMP during beneficial and commensal associations. This review describes the known roles of c-di-GMP in regulating phenotypes that contribute to host colonization, with a focus on knowledge gaps and future prospects for examining c-di-GMP during beneficial colonization. [ABSTRACT FROM AUTHOR]

Widespread phytochrome photoreceptors use photoisomerization of linear tetrapyrrole (bilin) chromophores to measure the ratio of red to far-red light. Cyanobacteria also contain distantly related cyanobacteriochrome (CBCR) proteins that share the bilin-binding GAF domain of phytochromes but sense other colors of light. CBCR photocycles are extremely diverse, ranging from the near-UV to the near-IR. Photoisomerization of the bilin triggers photoconversion of the CBCR input, thereby modulating the biochemical signaling state of output domains such as histidine kinase bidomains that can interface with cellular signal transduction pathways. CBCRs thus can regulate several aspects of cyanobacterial photobiology, including phototaxis, metabolism of cyclic nucleotide second messengers, and optimization of the cyanobacterial light-harvesting apparatus. This review examines spectral tuning, photoconversion, and photobiology of CBCRs and recent developments in understanding their evolution and in applying them in synthetic biology. [ABSTRACT FROM AUTHOR]

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

A recent article in the journal MedComm discusses the discovery of S-adenosyl methionine (SAM)-AMP as a novel class of second messengers for antiviral signals in bacterial defense systems. The study focuses on the type III CRISPR-Cas system and its unique characteristics in RNA cleavage and RNA-activated DNA cleavage for antiviral defense. The researchers identified SAM-AMP as a signaling molecule and found that it plays a role in bacterial antiviral immune defense. This discovery expands our understanding of bacterial molecules and sheds light on the evolutionary relationship between prokaryotic and eukaryotic signaling pathways. [Extracted from the article]

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs. [ABSTRACT FROM AUTHOR]

Calcium ion (Ca2+) is a multifaceted signaling molecule that acts as an important second messenger. During the course of evolution, plants and animals have developed Ca2+ signaling in order to respond against diverse stimuli, to regulate a large number of physiological and developmental pathways. Our understanding of Ca2+ signaling and its components in physiological phenomena ranging from lower to higher organisms, and from single cell to multiple tissues has grown exponentially. The generation of Ca2+ transients or signatures for various stress factor is a well-known mechanism adopted in plant and animal systems. However, the decoding of such remarkable signatures is an uphill task and is always an interesting goal for the scientific community. In the past few decades, studies on the concentration and dynamics of intracellular Ca2+ are significantly increasing and have become a trend in modern biology. The advancement in approaches from Ca2+ binding dyes to in vivo Ca2+ imaging through the use of Ca2+ biosensors to achieve spatio-temporal resolution in micro and milliseconds range, provide us phenomenal opportunities to study live cell Ca2+ imaging or dynamics. Here, we describe the usage, improvement and advancement of Ca2+ based dyes, genetically encoded probes and sensors to achieve extraordinary Ca2+ imaging in plants and animals. [ABSTRACT FROM AUTHOR]

In adrenal fasciculata cells stimulated by ACTH, Ca2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca2+-containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca2+-free medium. ACTH rapidly increased the intracellular free Ca2+ concentration ([Ca2+]i) in the normal medium, but did not influence [Ca2+]i in the Ca2+-free medium, indicating that ACTH caused Ca2+ influx into the cells. ACTH-induced Ca2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca2+ channel blocker but not by a T-type, N-type, or P-type Ca2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca2+-free medium and slowly caused Ca2+ influx into the cells. These results strongly suggest that Ca2+, as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

Abstract Background Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. Results Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. Conclusion The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti.

Summary: The 3′,5′‐cyclic adenosine monophosphate (cAMP) is a versatile second messenger in many mammalian signaling pathways. However, its role in plants remains not well‐recognized. Recent discovery of adenylate cyclase (AC) activity for transport inhibitor response 1/auxin‐signaling F‐box proteins (TIR1/AFB) auxin receptors and the demonstration of its importance for canonical auxin signaling put plant cAMP research back into spotlight. This insight briefly summarizes the well‐established cAMP signaling pathways in mammalian cells and describes the turbulent and controversial history of plant cAMP research highlighting the major progress and the unresolved points. We also briefly review the current paradigm of auxin signaling to provide a background for the discussion on the AC activity of TIR1/AFB auxin receptors and its potential role in transcriptional auxin signaling as well as impact of these discoveries on plant cAMP research in general. [ABSTRACT FROM AUTHOR]

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

Background: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. Results: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. Conclusion: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti. [ABSTRACT FROM AUTHOR]

C-di-GMP is a bacterial second messenger with central role in biofilm formation. Spirochete bacteria from Leptospira genus present a wide diversity, with species of medical importance and environmental species, named as saprophytic. Leptospira form biofilms in the rat’s reservoir kidneys and in the environment. Here, we performed genomic analyses to identify enzymatic and effector c-di-GMP proteins in the saprophytic biofilm-forming species Leptospira biflexa serovar Patoc. We identified 40 proteins through local alignments. Amongst them, 16 proteins are potentially functional diguanylate cyclases, phosphodiesterases, or hybrid proteins. We also identified nine effectors, including PilZ proteins. Enrichment analyses suggested that c-di-GMP interacts with cAMP signaling system, CsrA system, and flagella assembly regulation during biofilm development of L. biflexa. Finally, we identified eight proteins in the pathogen Leptospira interrogans serovar Copenhageni that share high similarity with L. biflexa c-di-GMP-related proteins. This work revealed proteins related to c-di-GMP turnover and cellular response in Leptospira and their potential roles during biofilm development. [ABSTRACT FROM AUTHOR]

Cyclic-di-AMP (c-di-AMP) is an important secondary messenger molecule that plays a critical role in monitoring several important cellular processes, especially in several Gram-positive bacteria. In this study, we seek to unravel the physiological significance of the molecule c-di-AMP in Mycobacterium smegmatis under different conditions, using strains with altered c-di-AMP levels: c-di-AMP null mutant (ΔdisA) and a c-di-AMP over-expression mutant (Δpde). Our thorough analysis of the mutants revealed that the intracellular concentration of c-di-AMP could determine many basic phenotypes such as colony architecture, cell shape, cell size, membrane permeability etc. Additionally, it was shown to play a significant role in multiple stress adaptation pathways in the case of different DNA and membrane stresses. Our study also revealed how the biofilm phenotypes of M. smegmatis cells are altered with high intracellular c-di-AMP concentration. Next, we checked how c-di-AMP contributes to antibiotic resistance or susceptibility characteristics of M. smegmatis, which was followed by a detailed transcriptome profile analysis to reveal key genes and pathways such as translation, arginine biosynthesis, cell wall and plasma membrane are regulated by c-di-AMP in mycobacteria.

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons. [ABSTRACT FROM AUTHOR]

Cyclic guanosine 3',5'-monophosphate (cGMP) is a ubiquitous important second messenger involved in various physiological functions. Here, intracellular cGMP (cGMPi) was visualized in chemotactic Dictyostelium cells using the fluorescent probe, D-Green cGull. When wild-type cells were stimulated with a chemoattractant, fluorescence transiently increased, but guanylate cyclase-null cells did not show a change in fluorescence, suggesting that D-Green cGull is a reliable indicator of cGMPi. In the aggregation stage, the responses of cGMPi propagated in a wave-like fashion from the aggregation center. The oscillation of the cGMPi wave was synchronized almost in phase with those of other second messengers, such as the intracellular cAMP and Ca2+. The phases of these waves preceded those of the oscillations of actomyosin and cell velocity, suggesting that these second messengers are upstream of the actomyosin and chemotactic migration. An acute increase in cGMPi concentration released from membrane-permeable caged cGMP induced a transient shuttle of myosin II between the cytosol and cell cortex, suggesting a direct link between cGMP signaling and myosin II dynamics. [ABSTRACT FROM AUTHOR]

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger discovered in Bacillus subtilis and involved in potassium homeostasis, cell wall maintenance and/or DNA stress response. As the role of c-di-AMP has been mostly studied in Firmicutes, we sought to increase the understanding of its role in Actinobacteria, namely in Corynebacterium glutamicum. This organism is a well-known industrial production host and a model organism for pathogens, such as C. diphtheriae or Mycobacterium tuberculosis. Here, we identify and analyze the minimal set of two C. glutamicum enzymes, the diadenylate cyclase DisA and the phosphodiesterase PdeA, responsible for c-di-AMP metabolism. DisA synthesizes c-di-AMP from two molecules of ATP, whereas PdeA degrades c-di-AMP, as well as the linear degradation intermediate phosphoadenylyl-(3′→5′)-adenosine (pApA) to two molecules of AMP. Here, we show that a ydaO/kimA-type c-di-AMP-dependent riboswitch controls the expression of the strictly regulated cell wall peptidase gene nlpC in C. glutamicum. In contrast to previously described members of the ydaO/kimA-type riboswitches, our results suggest that the C. glutamicum nlpC riboswitch likely affects the translation instead of the transcription of its downstream gene. Although strongly regulated by different mechanisms, we show that the absence of nlpC, the first known regulatory target of c-di-AMP in C. glutamicum, is not detrimental for this organism under the tested conditions. [ABSTRACT FROM AUTHOR]

To counter pathogen invasion, plants have evolved a large number of immune receptors, including membrane-resident pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat receptors (NLRs). Our knowledge about PRR and NLR signaling mechanisms has expanded significantly over the past few years. Plant NLRs form multi-protein complexes called resistosomes in response to pathogen effectors, and the signaling mediated by NLR resistosomes converges on Ca2+-permeable channels. Ca2+-permeable channels important for PRR signaling have also been identified. These findings highlight a crucial role of Ca2+ in triggering plant immune signaling. In this review, we first discuss the structural and biochemical mechanisms of non-canonical NLR Ca2+ channels and then summarize our knowledge about immune-related Ca2+-permeable channels and their roles in PRR and NLR signaling. We also discuss the potential role of Ca2+ in the intricate interaction between PRR and NLR signaling. Plant NLRs form resistosomes in response to pathogen effectors to initiate immune signaling. In this review, Wang et al. summarize recent findings concerning NLR resistosomes and discuss biochemical and structural mechanisms of how signaling mediated by different resistosomes converges on Ca2+ pathways. The potential roles of Ca2+ in mutual potentiation of NLR signaling and the signaling mediated by membrane-resident immune receptors are also discussed. [ABSTRACT FROM AUTHOR]

The dinucleotide cyclic di-AMP (c-di-AMP) is synthesized as a second messenger in the Gram-positive model bacterium Bacillus subtilis as well as in many bacteria and archaea. Bacillus subtilis possesses three diadenylate cyclases and two phosphodiesterases that synthesize and degrade the molecule, respectively. Among the second messengers, c-di-AMP is unique since it is essential for B. subtilis on the one hand but toxic upon accumulation on the other. This role as an "essential poison" is related to the function of c-di-AMP in the control of potassium homeostasis. C-di-AMP inhibits the expression and activity of potassium uptake systems by binding to riboswitches and transporters and activates the activity of potassium exporters. In this way, c-di-AMP allows the adjustment of uptake and export systems to achieve a balanced intracellular potassium concentration. C-di-AMP also binds to two dedicated signal transduction proteins, DarA and DarB. Both proteins seem to interact with other proteins in their apo state, i.e. in the absence of c-di-AMP. For DarB, the (p)ppGpp synthetase/hydrolase Rel and the pyruvate carboxylase PycA have been identified as targets. The interactions trigger the synthesis of the alarmone (p)ppGpp and of the acceptor molecule for the citric acid cycle, oxaloacetate, respectively. In the absence of c-di-AMP, many amino acids inhibit the growth of B. subtilis. This feature can be used to identify novel players in amino acid homeostasis. In this review, we discuss the different functions of c-di-AMP and their physiological relevance. [ABSTRACT FROM AUTHOR]

For many years the surprising multiplicity, signal input diversity, and output specificity of c-di-GMP signaling proteins has intrigued researchers studying bacterial second messengers. How can several signaling pathways act in parallel to produce specific outputs despite relying on the same diffusible second messenger maintained at a certain global cellular concentration? Such high specificity and flexibility arise from combining modes of local and global c-di-GMP signaling in complex signaling networks. Local c-di-GMP signaling can be experimentally shown by three criteria being met: (i) highly specific knockout phenotypes for particular c-di-GMP-related enzymes, (ii) actual cellular c-di-GMP levels that remain unchanged by such mutations and/or below the Kd's of the relevant c-di-GMP-binding effectors, and (iii) direct interactions between the signaling proteins involved. Here, we discuss the rationale behind these criteria and present well-studied examples of local c-di-GMP signaling in Escherichia coli and Pseudomonas. Relatively simple systems just colocalize a local source and/or a local sink for c-di-GMP, i.e. a diguanylate cyclase (DGC) and/or a specific phosphodiesterase (PDE), respectively, with a c-di-GMP-binding effector/target system. More complex systems also make use of regulatory protein interactions, e.g. when a "trigger PDE" responds to locally provided c-di-GMP, and thereby serves as a c-di-GMP-sensing effector that directly controls a target's activity, or when a c-di-GMP-binding effector recruits and directly activates its own "private" DGC. Finally, we provide an outlook into how cells can combine local and global signaling modes of c-di-GMP and possibly integrate those into other signaling nucleotides networks. [ABSTRACT FROM AUTHOR]

Second messengers transfer signals from changing intra- and extracellular conditions to a cellular response. Over the last few decades, several nucleotide-based second messengers have been identified and characterized in especially bacteria and eukaryotes. Also in archaea, several nucleotide-based second messengers have been identified. This review will summarize our understanding of nucleotide-based second messengers in archaea. For some of the nucleotide-based second messengers, like cyclic di-AMP and cyclic oligoadenylates, their roles in archaea have become clear. Cyclic di-AMP plays a similar role in osmoregulation in euryarchaea as in bacteria, and cyclic oligoadenylates are important in the Type III CRISPR–Cas response to activate CRISPR ancillary proteins involved in antiviral defense. Other putative nucleotide-based second messengers, like 3′,5′- and 2′,3′-cyclic mononucleotides and adenine dinucleotides, have been identified in archaea, but their synthesis and degradation pathways, as well as their functions as secondary messengers, still remain to be demonstrated. In contrast, 3′-3′-cGAMP has not yet been identified in archaea, but the enzymes required to synthesize 3′-3′-cGAMP have been found in several euryarchaeotes. Finally, the widely distributed bacterial second messengers, cyclic diguanosine monophosphate and guanosine (penta-)/tetraphosphate, do not appear to be present in archaea. [ABSTRACT FROM AUTHOR]

The alarmones and second messengers (p)ppGpp are important for the cellular response to amino acid starvation. Although the stringent response is present in many bacteria, the targets and functions of (p)ppGpp can differ between species, and our knowledge of (p)ppGpp targets is constantly expanding. Recently, it was demonstrated that these alarmones are also part of the heat shock response in Bacillus subtilis and that there is a functional overlap with the oxidative and heat stress transcriptional regulator Spx. Here, the (p)ppGpp second messenger alarmones allow the fast stress-induced downregulation of translation while Spx inhibits the further expression of translation-related genes to lower the load on the protein quality control system, while the chaperone and protease expression is induced. In this review, we discuss the role of (p)ppGpp and its intricate connections in the complex network of stress sensing, heat shock response, and adaptation in B. subtilis cells. [ABSTRACT FROM AUTHOR]

Cyclic di-AMP is an emerging second messenger that is synthesized by many archaea and bacteria, including the Gram-positive pathogenic bacterium Listeria monocytogenes. Listeria monocytogenes played a crucial role in elucidating the essential function of c-di-AMP, thereby becoming a model system for studying c-di-AMP metabolism and the influence of the nucleotide on cell physiology. c-di-AMP is synthesized by a diadenylate cyclase and degraded by two phosphodiesterases. To date, eight c-di-AMP receptor proteins have been identified in L. monocytogenes , including one that indirectly controls the uptake of osmotically active peptides and thus the cellular turgor. The functions of two c-di-AMP-receptor proteins still need to be elucidated. Here, we provide an overview of c-di-AMP signalling in L. monocytogenes and highlight the main differences compared to the other established model systems in which c-di-AMP metabolism is investigated. Moreover, we discuss the most important questions that need to be answered to fully understand the role of c-di-AMP in osmoregulation and in the control of central metabolism. [ABSTRACT FROM AUTHOR]

Abstract Glucagon is a potent glucose‐elevating hormone that is secreted by pancreatic α‐cells. While well‐controlled glucagon secretion plays an important role in maintaining systemic glucose homeostasis and preventing hypoglycaemia, it is increasingly apparent that defects in the regulation of glucagon secretion contribute to impaired counter‐regulation and hyperglycaemia in diabetes. It has therefore been proposed that pharmacological interventions targeting glucagon secretion/signalling can have great potential in improving glycaemic control of patients with diabetes. However, despite decades of research, a consensus on the precise mechanisms of glucose regulation of glucagon secretion is yet to be reached. Second messengers are a group of small intracellular molecules that relay extracellular signals to the intracellular signalling cascade, modulating cellular functions. There is a growing body of evidence that second messengers, such as cAMP and Ca2+, play critical roles in α‐cell glucose‐sensing and glucagon secretion. In this review, we discuss the impact of second messengers on α‐cell electrical activity, intracellular Ca2+ dynamics and cell exocytosis. We highlight the possibility that the interaction between different second messengers may play a key role in the glucose‐regulation of glucagon secretion.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotidebased second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections. [ABSTRACT FROM AUTHOR]

Human malaria, caused by infection with Plasmodium parasites, remains one of the most important global public health problems, with the World Health Organization reporting more than 240 million cases and 600,000 deaths annually as of 2020 (World malaria report 2021). Our understanding of the biology of these parasites is critical for development of effective therapeutics and prophylactics, including both antimalarials and vaccines. Plasmodium is a protozoan organism that is intracellular for most of its life cycle. However, to complete its complex life cycle and to allow for both amplification and transmission, the parasite must egress out of the host cell in a highly regulated manner. This review discusses the major pathways and proteins involved in the egress events during the Plasmodium life cycle--merozoite and gametocyte egress out of red blood cells, sporozoite egress out of the oocyst, and merozoite egress out of the hepatocyte. The similarities, as well as the differences, between the various egress pathways of the parasite highlight both novel cell biology and potential therapeutic targets to arrest its life cycle. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) leads to significant economic losses in the poultry industry worldwide and restricts the development of the poultry industry. Oxidative stress, through the production of reactive oxygen species (ROS), damage iron‑sulfur (FeS) clusters, cysteine and methionine protein residues, and DNA, and then result in bacterial cells death. APEC has evolved a series of regulation systems to sense and quickly and appropriately respond to oxidative stress. Quorum sensing (QS), second messenger (SM), transcription factors (TFs), small regulatory RNAs (sRNAs), and two-component system (TCS) are important regulation systems ubiquitous in bacteria. It is of great significance to control APEC infection through investigating the molecular regulation mechanism on APEC adapting to oxidative stress. However, how the cross-talk among these regulation systems co-regulates transcription of oxidative stress-response genes in APEC has not been reported. This review suggests exploring connector proteins that co-regulate these regulation systems that co-activate transcription of oxidative stress-response genes to disrupt bacterial antioxidative defense mechanism in APEC, and then using these connector proteins as drug targets to control APEC infection. This review might contribute to illustrating the functional mechanism of APEC adapting to oxidative stress and exploring potential drug targets for the prevention and treatment of APEC infection., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

When challenged by starvation, bacterial organisms synthesize guanosine pentaphosphate and tetraphosphate, collectively denoted as (p)ppGpp, as second messengers to reprogram metabolism toward slower growth and enhanced stress tolerance. When starvation is alleviated, the RelA-SpoT Homolog (RSH) hydrolases downregulate (p)ppGpp, cleaving the 3'-diphosphate to produce GTP or GDP. Metazoan RSH hydrolases possess phosphatase activity responsible for converting cytoplasmic NADPH to NADH in mammalian cells. Inhibitor development for this family may therefore provide therapies to combat bacterial infection or metabolic dysregulation. Despite the availability of dozens of high-resolution structures, catalytic mechanisms of RSH hydrolases have remained poorly understood. All RSH hydrolases tightly bind a Mn 2+ near its active center, which is believed sufficient for hydrolase activity. In contrast to this notion, we demonstrate, using the (p)ppGpp hydrolase SpoT from Acinetobacter baumannii, that a second divalent cation, presumably a Mg 2+ under physiological conditions, is required for efficient catalysis. We also show that SpoT preferentially cleaves 3'-diphosphate over 3'-phosphate substrates, likely due to a key coordination between the β-phosphate and the second metal center. Metazoan RSH hydrolase replaces this β-phosphate with the side chain of an aspartate residue, thereby functioning as a phosphatase. We propose a binuclear metallohydrolase model where an invariant ED (Glu-Asp) diad, previously believed to activate the water nucleophile, instead coordinates to a Mg 2+ center. The refined molecular and evolutionary blueprint of RSH hydrolases will provide a more reliable foundation for the development of small-molecule inhibitors of this important enzyme family., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Zinc (Zn2+) is an essential micronutrient and the second most abundant trace metal in the human body. The important role that Zn2+ plays in hemostasis is exemplified by platelet-related bleeding phenotypes coinciding with dietary Zn2+ deficiency. These phenotypes are rectified upon Zn2+ supplementation. Labile (unbound) Zn2+ is present in the plasma at micromolar levels, but is also detected in atherosclerotic plaques, and released from platelet α granules. Therefore, it is likely that localized Zn2+ concentrations are higher at sites of thrombosis and hemostasis. Exogenous Zn2+ is a regulator of the hemostatic responses, with roles during coagulation and platelet activation. Extracellular Zn2+ gains access to the platelet cytosol and induces full platelet activation at high concentrations, and potentiates platelets to activation by conventional agonists at lower concentrations. Zn2+-induced platelet activation is dependent on PKC and integrin αIIbβ3, and is associated with tyrosine phosphorylation of platelet proteins. Agonist evoked platelet activation results in intracellular Zn2+ ([Zn2+]i) fluctuations that are sensitive to the platelet redox state. Increases in [Zn2+]i correlate with activation responses, including shape change, granule release, αIIbβ3 activation and phosphatidyl-serine exposure, consistent with a role as a second messenger. This review provides insight into the numerous demonstrated and potential roles for Zn2+ in platelet function during thrombosis and hemostasis, highlighting its increasing acceptance as an intracellular and extracellular platelet regulatory agent.

Abstract Background Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Results Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). Conclusions PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

Pseudomonas aeruginosa is capable of thriving in diverse environments due to its network of regulatory components for effective response to stress factors. The survival of the bacteria is also dependent on the ability to discriminate between the acquisition of beneficial and non-beneficial genetic materials via horizontal gene transfer (HGT). Thus, bacteria have evolved the CRISPR-Cas adaptive immune system for defense against the deleterious effect of phage infection and HGT. By using the transposon mutagenesis approach, we identified the virulence factor regulator (Vfr) as a key regulator of the type I-F CRISPR-Cas system in P. aeruginosa. We showed that Vfr influences the expression of the CRISPR-Cas system through two signaling pathways in response to changes in calcium levels. Under calcium-rich conditions, Vfr indirectly regulates the CRISPR-Cas system via modulation of the AHL-QS gene expression, which could be vital for defense against phage infection at high cell density. When encountering calcium deficiency, however, Vfr can directly regulate the CRISPR-Cas system via a cAMP-dependent pathway. Furthermore, we provide evidence that mutation of vfr reduces the CRISPR-Cas spacer acquisition and interference of HGT. The results from this study add to the regulatory network of factors controlling the CRISPR-Cas system in response to abiotic factors in the environment. The findings may facilitate the design of effective and reliable phage therapies against P. aeruginosa infections, as targeting Vfr could prevent the development of the CRISPR-Cas mediated phage resistance.

The GPR15 receptor is a G protein‐coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25–81) and a C‐terminal peptide fragment GPR15L(71–81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3′,5′‐monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs, Gq/11 and G12/13 is unclear. Here, GPR15L(25–81) and GPR15L(71–81) are used as pharmacological tool compounds to delineate the GPR15 receptor‐mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1, Gi2, Gi3, GoA, GoB and Gz). The Emax and activation rates of Gi1, Gi2, Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin. [ABSTRACT FROM AUTHOR]

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an argR-null mutant grows poorly with arginine as the sole carbon or nitrogen source and also displays increased biofilm formation and reduced surface motility. Modulation of cdi-GMP levels by exogenous arginine requires ArgR. The expression of certain biofilm matrix components, namely, the adhesin LapF and the exopolysaccharide Pea, as well as the diguanylate cyclase CfcR is influenced by ArgR, likely through the alternative sigma factor RpoS. Our data indicate the existence of a regulatory feedback loop between ArgR and c-di-GMP mediated by FleQ. [ABSTRACT FROM AUTHOR]

cADPR is a second messenger that releases Ca2+ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2′-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does. [ABSTRACT FROM AUTHOR]

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger discovered in Bacillus subtilis and involved in potassium homeostasis, cell wall maintenance and/or DNA stress response. As the role of c-di-AMP has been mostly studied in Firmicutes, we sought to increase the understanding of its role in Actinobacteria, namely in Corynebacterium glutamicum. This organism is a well-known industrial production host and a model organism for pathogens, such as C. diphtheriae or Mycobacterium tuberculosis. Here, we identify and analyze the minimal set of two C. glutamicum enzymes, the diadenylate cyclase DisA and the phosphodiesterase PdeA, responsible for c-di-AMP metabolism. DisA synthesizes c-di-AMP from two molecules of ATP, whereas PdeA degrades c-di-AMP, as well as the linear degradation intermediate phosphoadenylyl-(3′→5′)-adenosine (pApA) to two molecules of AMP. Here, we show that a ydaO/kimA-type c-di-AMP-dependent riboswitch controls the expression of the strictly regulated cell wall peptidase gene nlpC in C. glutamicum. In contrast to previously described members of the ydaO/kimA-type riboswitches, our results suggest that the C. glutamicum nlpC riboswitch likely affects the translation instead of the transcription of its downstream gene. Although strongly regulated by different mechanisms, we show that the absence of nlpC, the first known regulatory target of c-di-AMP in C. glutamicum, is not detrimental for this organism under the tested conditions.

MnSOD catalyzes superoxide dismutation via temperature-dependent 'fast cycle' and 'slow cycle' pathways. Higher temperature promotes the formation of the product inhibition complexes, and the slow cycle pathway becomes mainstream in the overall dismutation. Decreasing the temperature reversely induces the dismutation into the fast cycle pathway. Cold-induced activation is a specific response to temperature and is a conservative feature of all MnSODs from microorganisms to humans. Cold-activation is an adaptive response to low temperature rather than a passive adaptation to excess superoxide. The cold-activated activity of MnSOD significantly exceeds the steady-state level of superoxide in the cell. Cold-activation is a regulatory mechanism for the prompt release of hydrogen peroxide fluxes which may act as a second messenger involving the regulation of adaptive thermogenesis. MnSOD may be a catalytic thermoreceptor playing a central role in adaptive thermogenesis.