TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Putrescine and Its Metabolic Precursor Arginine Promote Biofilm and c-di-GMP Synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.

Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF.

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure-function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF-RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand-receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor-synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

CRRL269.

RATIONALE: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. OBJECTIVE: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. METHODS AND RESULTS: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. CONCLUSIONS: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.

Influence of the N-terminal segment and the PHY-tongue element on light-regulation in bacteriophytochromes.

Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes. Red light-sensing bacteriophytochromes are attractive targets for the development of innovative optogenetic tools because of their natural modularity of coupling with diverse functionalities and the natural availability of the light-absorbing biliverdin chromophore in animal tissues. However, a rational design of such tools is complicated by the poor understanding of molecular mechanisms of light signal transduction over long distances-from the site of photon absorption to the active site of downstream enzymatic effectors. Here we show how swapping structural elements between two bacteriophytochrome homologs provides additional insight into light signal integration and effector regulation, involving a fine-tuned interplay of important structural elements of the sensor, as well as the sensor-effector linker. Facilitated by the availability of structural information of inhibited and activated full-length structures of one of the two homologs (Idiomarina species A28L phytochrome-activated diguanylyl cyclase (IsPadC)) and characteristic differences in photoresponses of the two homologs, we identify an important cross-talk between the N-terminal segment, containing the covalent attachment site of the chromophore, and the PHY-tongue region. Moreover, we highlight how these elements influence the dynamic range of photoactivation and how activation can be improved to light/dark ratios of ∼800-fold by reducing basal dark-state activities at the same time as increasing conversion in the light state. This will enable future optimization of optogenetic tools aiming at a direct allosteric regulation of enzymatic effectors.

A novel p.(Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors.

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 µM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

Structure of the active GGEEF domain of a diguanylate cyclase from Vibrio cholerae.

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.

Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches.

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca2+-dependent mechanisms. In darkness, Ca2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca2+ extrusion via Na+/Ca2+, K+ exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca2+ levels in photoreceptor outer segment because of continuing Ca2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca2+-feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones.

Low-Dose Carbon Monoxide Inhibits Rhinovirus Replication in Human Alveolar and Airway Epithelial Cells.

Carbon monoxide (CO) and nitric oxide (NO) exhibit physiological properties that include the activation of guanylate cyclase. NO inhibits replication of rhinovirus (RV), a major cause of the common cold and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. However, the anti-rhinoviral effects of CO remain unclear. This study investigated whether the exogenous application of low-dose CO could inhibit RV replication in human alveolar and airway epithelial cells. A549 human lung carcinoma cells with alveolar epithelial features and primary cultures of human tracheal epithelial (HTE) cells were pretreated with CO (100 ppm) and infected with a major group RV, type 14 RV (RV14). CO exposure reduced RV14 titers in the supernatants and RV RNA levels in A549 and HTE cells. The treatment with a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, reversed the inhibitory effects of CO exposure on RV14 replication in A549 cells. Pretreatment of A549 cells with 8-Br-cGMP, a cell-permeable cGMP analog, caused the decrease in RV14 replication, while CO exposure increased cGMP production. CO exposure also increased the expression levels of interferon (IFN)-γ mRNA and protein. In contrast, pretreatment with CO did not increase DNA fragmentation and did not reduce the expression of intercellular adhesion molecule-1, the RV14 receptor, or the number of acidic endosomes, through which RV RNA enters the cytoplasm. These findings suggest that low-dose CO may decrease RV14 replication in alveolar and airway epithelial cells. IFN-γ production, which is induced by CO exposure via guanylate cyclase activation-mediated cGMP production, may be involved in RV14 replication inhibition.

Role of Cyclic di-GMP in the Bacterial Virulence and Evasion of the Plant Immunity.

Plant pathogenic bacteria are responsible for the loss of hundreds of millions of dollars each year, impacting a wide range of economically relevant agricultural crops. The plant immune system detects conserved bacterial molecules and deploys an arsenal of effective defense measures at different levels; however, during compatible interactions, some pathogenic bacteria suppress and manipulate the host immunity and colonize and infect the plant host. Different bacteria employ similar strategies to circumvent plant innate immunity, while other tactics are specific to certain bacterial species. Recent studies have highlighted the secondary messenger c-di-GMP as a key molecule in the transmission of environmental cues in an intracellular regulatory network that controls virulence traits in many plant pathogenic bacteria. In this review, we focus on the recent knowledge of the molecular basis of c-di-GMP signaling mechanisms that promote or prevent the evasion of bacterial phytopathogens from the plant immune system. This review will highlight the considerable diversity of mechanisms evolved in plant-associated bacteria to elude plant immunity.

New Pharmacological Strategies to Increase cGMP.

The intracellular nucleotide cyclic guanosine monophosphate (cGMP) is found in many human organ tissues. Its concentration increases in response to the activation of receptor enzymes called guanylyl cyclases (GCs). Different ligands bind GCs, generating the second messenger cGMP, which in turn leads to a variety of biological actions. A deficit or dysfunction of this pathway at the cardiac, vascular, and renal levels manifests in cardiovascular diseases such as heart failure, arterial hypertension, and pulmonary arterial hypertension. An impairment of the cGMP pathway also may be involved in the pathogenesis of obesity as well as dementia. Therefore, agents enhancing the generation of cGMP for the treatment of these conditions have been intensively studied. Some have already been approved, and others are currently under investigation. This review discusses the potential of novel drugs directly or indirectly targeting cGMP as well as the progress of research to date.

Measurement of cGMP-generating and -degrading activities and cGMP levels in cells and tissues: Focus on FRET-based cGMP indicators.

The intracellular messenger molecule cGMP has an established function in the regulation of numerous physiological events. Yet for the identification of further biological cGMP-mediated functions, precise information whether a cGMP response exists in a certain cell type or tissue is mandatory. In this review, the techniques to measure cGMP i.e. cGMP-formation, -degradation or levels are outlined and discussed. As a superior method to measure cGMP, the article focusses on FRET-based cGMP indicators, describes the different cGMP indicators and discusses their advantages and drawbacks. Finally, the successful applications of these cGMP indicators to measure cGMP responses in cells and tissues are outlined and summarized. Hopefully, with the availability of the FRET-based cGMP indicators, the knowledge about the cGMP responses in special cells or tissues is going to increase thereby allowing to assess further cGMP-mediated functional responses and possibly to address their pathophysiology with the available guanylyl cyclase activators, stimulators and PDE inhibitors.

In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-ß and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

Cardiac unloading by LVAD support differentially influences components of the cGMP-PKG signaling pathway in ischemic and dilated cardiomyopathy.

Implantation of left ventricular assist devices (LVADs) as bridge to transplant in end-stage heart failure allows for analyzing reverse remodeling processes of the supported heart. Whether this therapy influences the cGMP-PKG signaling pathway, which is currently under thorough investigation for developing new heart failure therapeutics, is unknown. In fourteen end-stage heart failure patients (8 with dilated cardiomyopathy, DCM; 6 with ischemic cardiomyopathy, ICM) tissue specimens of left ventricles were collected at LVAD implantation and afterwards at receiver heart explantation, respectively. Then the expressions of key components of the cGMP-PKG signaling pathway were determined by polymerase chain reaction (ANP; BNP; natriuretic peptide receptor A, NPR-A; natriuretic peptide receptor C, NPR-C; neprilysin; NOS3; soluble guanylyl cyclase, sGC; PDE5; cGMP-dependent protein kinase G, PKG) and enzyme-linked immunosorbent assay (cGMP), respectively. Patients were predominantly male, 52 ± 10 years old, were receiving recommended heart failure therapy, and had their donor organ implanted after 351 ± 317 days of LVAD support. Except for more DCM patients with ICD therapy, no significant differences were detected between ICM and DCM, which also applies to the expression of cGMP-PKG pathway components at baseline. After LVAD support, ANP, NPR-C, and cGMP were significantly down-regulated and neprilysin, PDE5, and PKG I expressions were reduced with borderline significance in DCM, but not in ICM patients. Multiple significant correlations were found for expression differences (i.e., expression at LVAD implantation minus expression at heart transplantation) both in DCM and ICM, even though there was a closer connection between the NO and NP side of the cGMP-PKG pathway in DCM patients. Furthermore, duration of LVAD support negatively correlated with expression differences of PKG I, PDE5, and sGC in ICM, but not in DCM. Originating from the same activation level at LVAD implantation, cardiac unloading significantly alters key components of the cGMP-PKG pathway in DCM, but not in ICM patients. This etiology-specific regulation should be considered when analyzing therapeutic interventions with effects on this signaling pathway.

Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation.

We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3',5'-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.

The role of the globin-coupled sensor YddV in a mature E. coli biofilm population.

Biofilm-associated infections are hard to treat because of their high antibiotic resistance and the presence of a very persistent subpopulation of bacteria. The second messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) plays a very important role in this biofilm physiology. Here, we evaluated the role of YddV, an enzyme with a c-di-GMP synthesis function, in the formation and maturation of Escherichia coli biofilms. Our results suggest that YddV stimulates biofilm growth via its role in the production of c-di-GMP and this likely by influencing the production of matrix (e.g. poly-N-acetylglucosamine (PGA)). However, lowering the YddV expression did not alter the biofilm formation since there was no significant difference between the biofilm phenotypes of WT E. coli and YddV-knockout bacteria. Additionally, YddV expression had no significant influence on the amount of persister cells within the biofilm population, questioning the use of YddV as therapeutic target.

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation.

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signalling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.