Nucleotides as Bacterial Second Messengers.

In addition to comprising monomers of nucleic acids, nucleotides have signaling functions and act as second messengers in both prokaryotic and eukaryotic cells. The most common example is cyclic AMP (cAMP). Nucleotide signaling is a focus of great interest in bacteria. Cyclic di-AMP (c-di-AMP), cAMP, and cyclic di-GMP (c-di-GMP) participate in biological events such as bacterial growth, biofilm formation, sporulation, cell differentiation, motility, and virulence. Moreover, the cyclic-di-nucleotides (c-di-nucleotides) produced in pathogenic intracellular bacteria can affect eukaryotic host cells to allow for infection. On the other hand, non-cyclic nucleotide molecules pppGpp and ppGpp are alarmones involved in regulating the bacterial response to nutritional stress; they are also considered second messengers. These second messengers can potentially be used as therapeutic agents because of their immunological functions on eukaryotic cells. In this review, the role of c-di-nucleotides and cAMP as second messengers in different bacterial processes is addressed.

Control of bacterial second messenger signaling and motility by heme-based direct oxygen-sensing proteins.

Bacteria sense and respond to their environment, allowing them to maximize their survival and growth under changing conditions, such as oxygen levels. Direct oxygen-sensing proteins allow bacteria to rapidly sense concentration changes and adapt by regulating signaling pathways and/or cellular machinery. Recent work has identified roles for direct oxygen-sensing proteins in controlling second messenger levels and motility machinery, as well as effects on biofilm formation, virulence, and motility. In this review, we discuss recent progress in understanding O2-dependent regulation of cyclic di-GMP signaling and motility and highlight the emerging importance in controlling bacterial physiology and behavior.

Crosstalk between (p)ppGpp and other nucleotide second messengers.

In response to environmental cues, bacteria produce intracellular nucleotide messengers to regulate a wide variety of cellular processes and physiology. Studies on individual nucleotide messengers, such as (p)ppGpp or cyclic (di)nucleotides, have established their respective regulatory themes. As research on nucleotide signaling networks expands, recent studies have begun to uncover various crosstalk mechanisms between (p)ppGpp and other nucleotide messengers, including signal conversion, allosteric regulation, and target competition. The multiple layers of crosstalk implicate that (p)ppGpp is intricately linked to different nucleotide signaling pathways. From a physiological perspective, (p)ppGpp crosstalk enables fine-tuning and feedback regulation with other nucleotide messengers to achieve optimal adaptation.

Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

Cyclic-di-AMP signalling in lactic acid bacteria.

Cyclic dimeric adenosine monophosphate (cyclic-di-AMP) is a nucleotide second messenger present in Gram-positive bacteria, Gram-negative bacteria and some Archaea. The intracellular concentration of cyclic-di-AMP is adjusted in response to environmental and cellular cues, primarily through the activities of synthesis and degradation enzymes. It performs its role by binding to protein and riboswitch receptors, many of which contribute to osmoregulation. Imbalances in cyclic-di-AMP can lead to pleiotropic phenotypes, affecting aspects such as growth, biofilm formation, virulence, and resistance to osmotic, acid, and antibiotic stressors. This review focuses on cyclic-di-AMP signalling in lactic acid bacteria (LAB) incorporating recent experimental discoveries and presenting a genomic analysis of signalling components from a variety of LAB, including those found in food, and commensal, probiotic, and pathogenic species. All LAB possess enzymes for the synthesis and degradation of cyclic-di-AMP, but are highly variable with regards to the receptors they possess. Studies in Lactococcus and Streptococcus have revealed a conserved function for cyclic-di-AMP in inhibiting the transport of potassium and glycine betaine, either through direct binding to transporters or to a transcriptional regulator. Structural analysis of several cyclic-di-AMP receptors from LAB has also provided insights into how this nucleotide exerts its influence.

Segregation of Ca2+ signaling in olfactory signal transduction.

Olfactory signal transduction is conducted through a cAMP-mediated second messenger cascade. The cytoplasmic Ca2+ concentration increases through the opening of CNG channels, a phenomenon that underlies two major functions, namely, signal boosting and olfactory adaptation. Signal boosting is achieved by an additional opening of the Ca2+-activated Cl- channel whereas adaptation is regulated by Ca2+ feedback to the CNG channel. Thus, the influx of Ca2+ and the resultant increase in cytoplasmic Ca2+ levels play seemingly opposing effects: increasing the current while reducing the current through adaptation. The two functions could be interpreted as compensating for each other. However, in real cells, both functions should be segregated. Ca2+ dynamics in olfactory cilia need to be directly measured, but technical difficulties accompanying the thin structure of olfactory cilia have prevented systematic analyses. In this study, using a combination of electrophysiology, local photolysis of caged cAMP, and Ca2+ imaging, we found that free Ca2+ in the local ciliary cytoplasm decreased along with a reduction in the current containing Ca2+-activated Cl- components returning to the basal level, whereas Ca2+-dependent adaptation persisted for a longer period. The activity of Cl- channels is highly likely to be regulated by the free Ca2+ that is present only immediately after the influx through the CNG channel, and an exclusive interaction between Ca2+ and Ca2+-binding proteins that mediate the adaptation may modulate the adaptation lifetime.

Regulation of cardiac function by cAMP nanodomains.

Cyclic adenosine monophosphate (cAMP) is a diffusible intracellular second messenger that plays a key role in the regulation of cardiac function. In response to the release of catecholamines from sympathetic terminals, cAMP modulates heart rate and the strength of contraction and ease of relaxation of each heartbeat. At the same time, cAMP is involved in the response to a multitude of other hormones and neurotransmitters. A sophisticated network of regulatory mechanisms controls the temporal and spatial propagation of cAMP, resulting in the generation of signaling nanodomains that enable the second messenger to match each extracellular stimulus with the appropriate cellular response. Multiple proteins contribute to this spatiotemporal regulation, including the cAMP-hydrolyzing phosphodiesterases (PDEs). By breaking down cAMP to a different extent at different locations, these enzymes generate subcellular cAMP gradients. As a result, only a subset of the downstream effectors is activated and a specific response is executed. Dysregulation of cAMP compartmentalization has been observed in cardiovascular diseases, highlighting the importance of appropriate control of local cAMP signaling. Current research is unveiling the molecular organization underpinning cAMP compartmentalization, providing original insight into the physiology of cardiac myocytes and the alteration associated with disease, with the potential to uncover novel therapeutic targets. Here, we present an overview of the mechanisms that are currently understood to be involved in generating cAMP nanodomains and we highlight the questions that remain to be answered.

Controlling Biofilm Development Through Cyclic di-GMP Signaling.

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date summary of c-di-GMP pathways connected to biofilm formation by the opportunistic pathogen P. aeruginosa. Emphasis will be on the timing of c-di-GMP production over the course of biofilm formation, to highlight non-uniform and hierarchical increases in c-di-GMP levels, as well as biofilm growth conditions that do not conform with our current model of c-di-GMP.

Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE.

cAMP is a ubiquitous second messenger involved in the regulation of diverse cellular processes. Spatiotemporal regulation of cAMP through compartmentalization within various subcellular microdomains is essential to ensure specific signaling. In the following protocol, we describe a method for directly visualizing signaling dynamics within cAMP microdomains using fluorescent sensors targeted to endogenous proteins (FluoSTEPs). Instead of overexpressing a biosensor-tagged protein of interest to target a microdomain, FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE) utilizes spontaneously complementing split GFP and CRISPR-Cas9 genome editing to localize a FRET-based cAMP biosensor to an endogenously expressed protein of interest. Utilizing this approach, FluoSTEP-ICUE can be used to measure cAMP levels within endogenous signaling compartments, thus providing a powerful tool for studying the spatiotemporal regulation of cAMP signaling.

The role of lipid second messengers in aldosterone synthesis and secretion.

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.

Signaling events that occur when cells of Escherichia coli encounter a glass surface.

Bacterial cells interact with solid surfaces and change their lifestyle from single free-swimming cells to sessile communal structures (biofilms). Cyclic di-guanosine monophosphate (c-di-GMP) is central to this process, yet we lack tools for direct dynamic visualization of c-di-GMP in single cells. Here, we developed a fluorescent protein-based c-di-GMP-sensing system for Escherichia coli that allowed us to visualize initial signaling events and assess the role played by the flagellar motor. The sensor was pH sensitive, and the events that appeared on a seconds' timescale were alkaline spikes in the intracellular pH. These spikes were not apparent when signals from different cells were averaged. Instead, a signal appeared on a minutes' timescale that proved to be due to an increase in intracellular c-di-GMP. This increase, but not the alkaline spikes, depended upon a functional flagellar motor. The kinetics and the amplitude of both the pH and c-di-GMP responses displayed cell-to-cell variability indicative of the distinct ways the cells approached and interacted with the surface. The energetic status of a cell can modulate these events. In particular, the alkaline spikes displayed an oscillatory behavior and the c-di-GMP increase was modest in the presence of glucose.

Phosphodiesterases and Compartmentation of cAMP and cGMP Signaling in Regulation of Cardiac Contractility in Normal and Failing Hearts.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through ß-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

Roles of the second messenger c-di-GMP in bacteria: Focusing on the topics of flagellar regulation and Vibrio spp.

Typical second messengers include cyclic AMP (cAMP), cyclic GMP (cGMP), and inositol phosphate. In bacteria, cyclic diguanylate (c-di-GMP), which is not used in animals, is widely used as a second messenger for environmental responses. Initially found as a regulator of cellulose synthesis, this small molecule is known to be widely present in bacteria. A wide variety of synthesis and degradation enzymes for c-di-GMP exist, and the activities of effector proteins are regulated by changing the cellular c-di-GMP concentration in response to the environment. It has been shown well that c-di-GMP plays an essential role in pathogenic cycle and is involved in flagellar motility in Vibrio cholerae. In this review, we aim to explain the direct or indirect regulatory mechanisms of c-di-GMP in bacteria, focusing on the study of c-di-GMP in Vibrio spp. and in flagella, which are our research subjects.

Second messenger signaling in Clostridioides difficile.

Small, diffusible second messenger molecules transmit information about extracellular conditions to intracellular machinery in order to influence transcription, translation, and metabolism. The enteropathogenic bacterium Clostridioides difficile coordinates its response to a dynamic and hostile environment via nucleotide second messengers. While riboswitch-mediated cyclic diguanylate regulation has been extensively characterized in C. difficile, signaling by cyclic diadenylate and by guanosine alarmones has only recently been confirmed in this organism. This review summarizes the current knowledge of how nucleotide second messenger signaling regulates physiological processes in C. difficile.

Engaging biological oscillators through second messenger pathways permits emergence of a robust gastric slow-wave during peristalsis.

Peristalsis, the coordinated contraction-relaxation of the muscles of the stomach is important for normal gastric motility and is impaired in motility disorders. Coordinated electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICC) and smooth muscle (SM) cells of the stomach wall as a slow-wave, underly peristalsis. Normally, the gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. Understanding of the integrative role of neurotransmission and intercellular coupling in the propagation of an entrained gastric slow-wave, important for understanding motility disorders, however, remains incomplete. Using a computational framework constituted of a novel gastric motility network (GMN) model we address the hypothesis that engaging biological oscillators (i.e., ICCs) by constitutive gap junction coupling mechanisms and enteric neural innervation activated signals can confer a robust entrained gastric slow-wave. We demonstrate that while a decreasing enteric neural innervation gradient that modulates the intracellular IP3 concentration in the ICCs can guide the aboral slow-wave propagation essential for peristalsis, engaging ICCs by recruiting the exchange of second messengers (inositol trisphosphate (IP3) and Ca2+) ensures a robust entrained longitudinal slow-wave, even in the presence of biological variability in electrical coupling strengths. Our GMN with the distinct intercellular coupling in conjunction with the intracellular feedback pathways and a rostrocaudal enteric neural innervation gradient allows gastric slow waves to oscillate with a moderate range of frequencies and to propagate with a broad range of velocities, thus preventing decoupling observed in motility disorders. Overall, the findings provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach, offer directions for future experiments and theoretical work, and can potentially aid in the design of new interventional pharmacological and neuromodulation device treatments for addressing gastric motility disorders.

The c-di-AMP signaling system influences stress tolerance and biofilm formation of Streptococcus mitis.

Streptococcus mitis is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization, S. mitis must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic-di-AMP (c-di-AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic-di-AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in S. mitis, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c-di-AMP. In this study, we utilized S. mitis deletion mutants of cdaA, pde1, and pde2 to analyze the role of c-di-AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δpde1 mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of cdaA increases auto-aggregation of S. mitis but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of pde1 or pde2 reduced the tolerance to ciprofloxacin, and UV radiation and the Δpde1 mutant was more susceptible to Triton X-100, indicating a role for c-di-AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δpde2 mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c-di-AMP signaling in cellular processes important for colonization of the mouth.

Endoplasmic Reticulum-Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling.

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Bacterial cyclic diguanylate signaling networks sense temperature.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

Link between Lipid Second Messengers and Osmotic Stress in Plants.

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.

Cyclic di-GMP sensing histidine kinase PdtaS controls mycobacterial adaptation to carbon sources.

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.