Quantitative input-output dynamics of a c-di-GMP signal-transduction cascade in Vibrio cholerae

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second-messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which in turn, drives alterations specifically to biofilm gene expression. A longstanding question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

Transmembrane signalling by a bionic receptor: biological input and output, chemical mechanism of signal transduction

Signal transduction through sealed biological membranes is among the most important evolutionary achievements. Herein, we focus on the development of artificial signal transduction mechanisms and engineer a bionic receptor with capacity of transduction of biological signals across biological membranes using tools of chemistry. The bionic receptor described in this work exhibits similarity with the natural counterpart in the most essential characteristics: in having an exofacial ligand for signal capture, in being membrane anchored, and in featuring a releasable secondary messenger molecule, which performs enzyme activation in the endo volume. The main difference with the natural receptors is that signal transduction across the lipid bilayer was performed using the tools of organic chemistry, namely a self-immolative linker. The highest novelty of our work is that the artificial signalling cascade designed herein achieved transmembrane activation of enzymatic activity, as is the hallmark of activity by natural signalling receptors.

A nucleus targetable fluorescent probe for ratiometric imaging of endogenous NO in living cells and zebrafishes

Nitric oxide (NO) is an important cellular messenger molecule in the cardiovascular, nervous and immune systems. Real-time monitoring of NO activity in specific organelles of live cells is important to...

Cyclic di-AMP Signaling in Bacteria

The second messenger molecule cyclic di-AMP (c-di-AMP) is formed by many bacteria and archaea. In many species that produce c-di-AMP, this second messenger is essential for viability on rich medium. Recent research has demonstrated that c-di-AMP binds to a large number of proteins and riboswitches, which are often involved in potassium and osmotic homeostasis. c-di-AMP becomes dispensable if the bacteria are cultivated on minimal media with low concentrations of osmotically active compounds. Thus, the essentiality of c-di-AMP does not result from an interaction with a single essential target but rather from the multilevel control of complex homeostatic processes. This review summarizes current knowledge on the homeostasis of c-di-AMP and its function(s) in the control of cellular processes.

cGMP: a unique 2nd messenger molecule – recent developments in cGMP research and development

Cyclic guanosine monophosphate (cGMP) is a unique second messenger molecule formed in different cell types and tissues. cGMP targets a variety of downstream effector molecules and, thus, elicits a very broad variety of cellular effects. Its production is triggered by stimulation of either soluble guanylyl cyclase (sGC) or particulate guanylyl cyclase (pGC); both enzymes exist in different isoforms. cGMP-induced effects are regulated by endogenous receptor ligands such as nitric oxide (NO) and natriuretic peptides (NPs). Depending on the distribution of sGC and pGC and the formation of ligands, this pathway regulates not only the cardiovascular system but also the kidney, lung, liver, and brain function; in addition, the cGMP pathway is involved in the pathogenesis of fibrosis, inflammation, or neurodegeneration and may also play a role in infectious diseases such as malaria. Moreover, new pharmacological approaches are being developed which target sGC- and pGC-dependent pathways for the treatment of various diseases. Therefore, it is of key interest to understand this pathway from scratch, beginning with the molecular basis of cGMP generation, the structure and function of both guanylyl cyclases and cGMP downstream targets; research efforts also focus on the subsequent signaling cascades, their potential crosstalk, and also the translational and, ultimately, the clinical implications of cGMP modulation. This review tries to summarize the contributions to the “9th International cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications” held in Mainz in 2019. Presented data will be discussed and extended also in light of recent landmark findings and ongoing activities in the field of preclinical and clinical cGMP research.

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

The Vc2 Cyclic di-GMP-Dependent Riboswitch of Vibrio cholerae Regulates Expression of an Upstream Putative Small RNA by Controlling RNA Stability

ABSTRACT Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream noncoding RNAs with 3′ ends located immediately downstream of the Vc2 riboswitch. Our results suggest these putative small RNAs (sRNAs) are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2. IMPORTANCE Riboswitches are typically RNA elements located in the 5′ untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or translation initiation. In this work, we report a novel mechanism of riboswitch-mediated gene regulation in Vibrio cholerae whereby a 3′ riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed putative sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structured RNA, we hypothesize this mechanism of gene regulation is widespread.

Ozone therapy in chronic diseases; a narrative review of the literature

Nitric oxide (NO) has various physiological and biochemical effects. In many biological systems of the body, NO acts as a messenger molecule via cyclic guanosine monophosphate (cGMP), which affects the body cells. NO is synthesized in the body from the L-arginine amino acid by the NO synthases enzyme. This enzyme consists of three major isoforms including neurotransmitter, endothelial and inductive types. According to the results of numerous studies, the administration of ozone as a complementary therapy of the diseases is a less complicated and cost-effective way. Over the past forty years, the results of ozone therapy have been satisfactory and without any problems. Ozone therapy affects various diseases. For example, in treatment for vascular diseases and some degenerative diseases, it has an ameliorative impact. Regarding kidney disease, still many experimental or clinical studies are necessary to find its improvement/anti-oxidative effect.

The Vc2 cyclic di-GMP dependent riboswitch ofVibrio choleraeregulates expression of an upstream small RNA by controlling RNA stability

SUMMARYCyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology ofVibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the genetfoY, functions as an off-switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression oftfoYhas not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream non-coding small RNAs (sRNA) with 3’-ends located immediately downstream of the Vc2 riboswitch. Our results suggest these sRNAs are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2.IMPORTANCERiboswitches are typically RNA elements located in the 5’ untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or the translation initiation. In this work, we report a novel mechanism of riboswitch mediated gene regulation inVibrio choleraewhereby a 3’ riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structure RNA, we hypothesize this mechanism of gene regulation could be widespread.

Cyclic di-GMP Regulates TfoY inVibrio choleraeTo Control Motility by both Transcriptional and Posttranscriptional Mechanisms

ABSTRACT3′,5′-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator inVibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. One such regulatory factor that may respond to c-di-GMP is the Vc2 c-di-GMP-binding riboswitch that is hypothesized to control the expression of the downstream putative transcription factor TfoY. Although much is known about the physical and structural properties of the Vc2 riboswitch aptamer, the nature of its expression and function inV. choleraehas not been investigated. Here, we show that Vc2 functions as an off switch to inhibit TfoY production at intermediate and high concentrations of c-di-GMP. At low c-di-GMP concentrations, TfoY production is induced to stimulate dispersive motility. We also observed increased transcription oftfoYat high intracellular concentrations of c-di-GMP, but this induction is independent of the Vc2 riboswitch and occurs via transcriptional control of promoters upstream oftfoYby the previously identified c-di-GMP dependent transcription factor VpsR. Our results show that TfoY is induced by c-di-GMP at both low and high intracellular concentrations of c-di-GMP via posttranscriptional and transcriptional mechanisms, respectively. This regulation contributes to the formation of three distinct c-di-GMP signaling states inV. cholerae.IMPORTANCEThe bacterial pathogenVibrio choleraemust transition between life in aquatic environmental reservoirs and life in the gastrointestinal tract. Biofilm formation and bacterial motility, and their control by the second messenger molecule c-di-GMP, play integral roles in this adaptation. Here, we define the third major mechanism by which c-di-GMP controls bacterial motility. This pathway utilizes a noncoding RNA element known as a riboswitch that, when bound to c-di-GMP, inhibits the expression of the transcription factor TfoY. TfoY production switchesV. choleraemotility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network ofV. choleraecan exist in at least three distinct states to regulate biofilm formation and motility.