Correction: Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1010219.].

Predicting butyrate- and propionate-forming bacteria of gut microbiota from sequencing data.

The bacteria-derived short-chain fatty acids (SCFAs) butyrate and propionate play important (distinct) roles in health and disease, and understanding the ecology of respective bacteria on a community-wide level is a top priority in microbiome research. Applying sequence data (metagenomics and 16S rRNA gene) to predict SCFAs production in vitro and in vivo, a clear split between butyrate- and propionate-forming bacteria was detected with only very few taxa exhibiting pathways for the production of both SCFAs. After in vitro growth of fecal communities from distinct donors (n = 8) on different substrates (n = 7), abundances of bacteria exhibiting pathways correlated with respective SCFA concentrations, in particular in the case of butyrate. For propionate, correlations were weaker, indicating that its production is less imprinted into the core metabolism compared with butyrate-forming bacteria. Longitudinal measurements in vivo (n = 5 time-points from 20 subjects) also revealed a correlation between abundances of pathway-carrying bacteria and concentrations of the two SCFAs. Additionally, lower bacterial cell concentrations, together with higher stool moisture, promoted overall bacterial activity (measured by flow cytometry and coverage patterns of metagenome-assembled genomes) that led to elevated SCFA concentrations with over-proportional levels of butyrate. Predictions on pathway abundances based on 16S rRNA gene data using our in-house database worked well, yielding similar results as metagenomic-based analyses. Our study indicates that stimulating growth of butyrate- and propionate-producing bacteria directly leads to more production of those compounds, which is governed by two functionally distinct bacterial groups facilitating the development of precision intervention strategies targeting either metabolite.

Vitamin A preserves cardiac energetic gene expression in a murine model of diet-induced obesity.

Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.NEW & NOTEWORTHY The relationship between vitamin-A status and the pathogenesis of diabetic cardiomyopathy has not been studied in detail. We assessed cardiac mitochondrial respiratory capacity, contractile function, and gene expression by RNA sequencing in a murine model of combined vitamin-A deficiency and diet-induced obesity. Our study identifies a role for vitamin A in preserving cardiac energetic gene expression that might attenuate subsequent development of mitochondrial and contractile dysfunction in diet-induced obesity.

Distinct Long- and Short-Term Adaptive Mechanisms in Pseudomonas aeruginosa.

Heterogeneous environments such as the chronically infected cystic fibrosis lung drive the diversification of Pseudomonas aeruginosa populations into, e.g., mucoid, alginate-overproducing isolates or small-colony variants (SCVs). In this study, we performed extensive genome and transcriptome profiling on a clinical SCV isolate that exhibited high cyclic diguanylate (c-di-GMP) levels and a mucoid phenotype. We observed a delayed, stepwise decrease of the high levels of c-di-GMP as well as alginate gene expression upon passaging the SCV under noninducing, rich medium growth conditions over 7 days. Upon prolonged passaging, this lagging reduction of the high c-di-GMP levels under noninducing planktonic conditions (reminiscent of a hysteretic response) was followed by a phenotypic switch to a large-colony morphology, which could be linked to mutations in the Gac/Rsm signaling pathway. Complementation of the Gac/Rsm signaling-negative large-colony variants with a functional GacSA system restored the SCV colony morphotype but was not able to restore the high c-di-GMP levels of the SCV. Our data thus suggest that expression of the SCV colony morphotype and modulation of c-di-GMP levels are genetically separable and follow different evolutionary paths. The delayed switching of c-di-GMP levels in response to fluctuating environmental conditions might provide a unique opportunity to include a time dimension to close the gap between short-term phenotypic and long-term genetic adaptation to biofilm-associated growth conditions. IMPORTANCE Extreme environments, such as those encountered during an infection process in the human host, make effective bacterial adaptation inevitable. While bacteria adapt individually by activating stress responses, long-term adaptation of bacterial communities to challenging conditions can be achieved via genetic fixation of favorable traits. In this study, we describe a two-pronged bacterial stress resistance strategy in the opportunistic pathogen Pseudomonas aeruginosa. We show that the production of adjusted elevated c-di-GMP levels, which drive protected biofilm-associated phenotypes in vivo, resembles a stable hysteretic response which prevents unwanted frequent switching. Cellular hysteresis might provide a link between individual adaptability and evolutionary adaptation to ensure the evolutionary persistence of host-adapted stress response strategies.

Itaconate Isomers in Bread.

The naturally occurring isomers itaconate, mesaconate and citraconate possess immunomodulatory, antioxidative and antimicrobial properties. However, it is not known whether they occur in commonly consumed human foods. Considering that they can arise as a result of heat conversion, we tested whether they occur in bread, representing a commonly consumed baked good. Using high-performance liquid chromatography−tandem mass spectrometry, we measured concentrations of the three isomers and their potential precursors, citrate and cis-aconitate, in unbaked sourdough and dough, and in crumb and crust of baked bread. All three isomers were detected at low concentrations (<20 pmol/mg dry weight) in sourdough, dough, crumb and crust. Concentrations of itaconate and citraconate were substantially higher in crust than in crumb of wheat and rye bread, and a modest increase in mesaconate was observed in crust of rye bread. In contrast, cis-aconitate concentrations were considerably lower in crust, which was consistent with the conversion of cis-aconitate to itaconate isomers due to higher temperature of the dough surface during baking. Based on data on the average consumption of bread and related baked goods in Germany, the daily intake of itaconate isomers was estimated to be roughly 7−20 µg. Thus, baked goods constitute a regular dietary source of low amounts of itaconate isomers. In order to enable studies on the impact of dietary intake of itaconate isomers on human health, their concentrations should be assessed in other foods that are subjected to high heating.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence.

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3',5')-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe-microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

miRNA-200b-A Potential Biomarker Identified in a Porcine Model of Cardiogenic Shock and Mechanical Unloading.

Background: Cardiogenic shock (CS) alters whole body metabolism and circulating biomarkers serve as prognostic markers in CS patients. Percutaneous ventricular assist devices (pVADs) unload the left ventricle by actively ejecting blood into the aorta. The goal of the present study was to identify alterations in circulating metabolites and transcripts in a large animal model that might serve as potential prognostic biomarkers in acute CS and additional left ventricular unloading by Impella ® pVAD support. Methods: CS was induced in a preclinical large animal model by injecting microspheres into the left coronary artery system in six pigs. After the induction of CS, mechanical pVAD support was implemented for 30 min total. Serum samples were collected under basal conditions, after the onset of CS, and following additional pVAD unloading. Circulating metabolites were determined by metabolomic analysis, circulating RNA entities by RNA sequencing. Results: CS and additional pVAD support alter the abundance of circulating metabolites involved in Aminoacyl-tRNA biosynthesis and amino acid metabolism. RNA sequencing revealed decreased abundance of the hypoxia sensitive miRNA-200b following the induction of CS, which was reversed following pVAD support. Conclusion: The hypoxamir miRNA-200b is a potential circulating marker that is repressed in CS and is restored following pVAD support. The early transcriptional response with increased miRNA-200b expression following only 30 min of pVAD support suggests that mechanical unloading alters whole body metabolism. Future studies are required to delineate the impact of serum miRNA-200b levels as a prognostic marker in patients with acute CS and pVAD unloading.

Thermosynechococcus switches the direction of phototaxis by a c-di-GMP-dependent process with high spatial resolution.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Cyanobacteria, like plants, grow by capturing energy from sunlight. But they have an advantage over their leafy counterparts: they can explore their environment to find the type of light that best suits their needs. These movements rely on hook-like structures, called type IV pili, which allow the cells to pull themselves forward. The pili are usually located at the opposite poles of a rod-shaped cell, allowing the bacteria to move along their longer axis. Yet, the molecular mechanisms that allow cyanobacteria to react to the light are poorly understood. To explore these processes in more detail, Nakane, Enomoto et al. started by shining coloured lights on the rod-shaped cyanobacteria Thermosynechococcus vulcanus. This revealed that the cells moved towards green light but reversed rapidly when blue light was added. The behaviour was disrupted when the genes for three light-sensing proteins were artificially switched off. These molecular players act by changing the levels of cyclic di-GMP, a signalling molecule that may interact with type IV pili. The experiments also showed that T. vulcanus cells were not only moving along their longer axis, but also at a right-angle. This observation contrasts with how other rod-shaped bacteria can explore their environment. A closer look revealed that the cyanobacteria could perform these movements by making asymmetrical adjustment to the way that pili at each pole were working. Further research is now needed to more finely dissect the molecular mechanisms which control this remarkable type of motion.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

GGDEF domain as spatial on-switch for a phosphodiesterase by interaction with landmark protein HubP.

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bimodal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here, we show that direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the FimV domain of the polar landmark protein HubP is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface-adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations, generated by differences in abundance and timing of polar appearance of PdeB, orchestrates the population behavior with respect to cell-surface interaction and environmental spreading.

Pseudomonas aeruginosa post-translational responses to elevated c-di-GMP levels.

C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

Octopamine drives honeybee thermogenesis.

In times of environmental change species have two options to survive: they either relocate to a new habitat or they adapt to the altered environment. Adaptation requires physiological plasticity and provides a selection benefit. In this regard, the Western honeybee (Apis mellifera) protrudes with its thermoregulatory capabilities, which enables a nearly worldwide distribution. Especially in the cold, shivering thermogenesis enables foraging as well as proper brood development and thus survival. In this study, we present octopamine signaling as a neurochemical prerequisite for honeybee thermogenesis: we were able to induce hypothermia by depleting octopamine in the flight muscles. Additionally, we could restore the ability to increase body temperature by administering octopamine. Thus, we conclude that octopamine signaling in the flight muscles is necessary for thermogenesis. Moreover, we show that these effects are mediated by ß octopamine receptors. The significance of our results is highlighted by the fact the respective receptor genes underlie enormous selective pressure due to adaptation to cold climates. Finally, octopamine signaling in the service of thermogenesis might be a key strategy to survive in a changing environment.

Staphylococcus aureus Multiplexes Death-Effector Deoxyribonucleosides to Neutralize Phagocytes.

Adenosine synthase A (AdsA) is a key virulence factor of Staphylococcus aureus, a dangerous microbe that causes fatal diseases in humans. Together with staphylococcal nuclease, AdsA generates deoxyadenosine (dAdo) from neutrophil extracellular DNA traps thereby igniting caspase-3-dependent cell death in host immune cells that aim at penetrating infectious foci. Powered by a multi-technological approach, we here illustrate that the enzymatic activity of AdsA in abscess-mimicking microenvironments is not restricted to the biogenesis of dAdo but rather comprises excessive biosynthesis of deoxyguanosine (dGuo), a cytotoxic deoxyribonucleoside generated by S. aureus to eradicate macrophages of human and animal origin. Based on a genome-wide CRISPR-Cas9 knock-out screen, we further demonstrate that dGuo-induced cytotoxicity in phagocytes involves targeting of the mammalian purine salvage pathway-apoptosis axis, a signaling cascade that is concomitantly stimulated by staphylococcal dAdo. Strikingly, synchronous targeting of this route by AdsA-derived dGuo and dAdo boosts macrophage cell death, indicating that S. aureus multiplexes death-effector deoxyribonucleosides to maximize intra-host survival. Overall, these data provide unique insights into the cunning lifestyle of a deadly pathogen and may help to design therapeutic intervention strategies to combat multidrug-resistant staphylococci.

Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways.

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius.

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3',5'-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3',5'-cAMP, cyclic guanosine monophosphate (3',5'-cGMP), 5'-phosphoadenylyl-3',5'-adenosine (5'-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2',3'-cyclic isomers of all four RNA building blocks (2',3'-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3',5'-cCMP), cyclic uridine monophosphate (3',5'-cUMP) and cyclic diadenosine monophosphate (3',5'-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3',5'-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

The two Pseudomonas aeruginosa DksA stringent response proteins are largely interchangeable at the whole transcriptome level and in the control of virulence-related traits.

The stringent response regulator DksA plays a key role in Gram negative bacteria adaptation to challenging environments. Intriguingly, the plant and human pathogen Pseudomonas aeruginosa is unique as it expresses two functional DksA paralogs: DksA1 and DksA2. However, the role of DksA2 in P. aeruginosa adaptive strategies has been poorly investigated so far. Here, RNA-Seq analysis and phenotypic assays showed that P. aeruginosa DksA1 and DksA2 proteins are largely interchangeable. Relative to wild type P. aeruginosa, transcription of 1779 genes was altered in a dksA1 dksA2 double mutant, and the wild type expression level of ≥90% of these genes was restored by in trans complementation with either dksA1 or dksA2. Interestingly, the expression of a small sub-set of genes seems to be preferentially or exclusively complemented by either dksA1 or dksA2. In addition, evidence has been provided that the DksA-dependent regulation of virulence genes expression is independent and hierarchically dominant over two major P. aeruginosa regulatory circuits, i.e., quorum sensing and cyclic-di-GMP signalling systems. Our findings support the prominent role of both DksA paralogs in P. aeruginosa environmental adaptation.

Minor pilins are involved in motility and natural competence in the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

Histamine can be Formed and Degraded in the Human and Mouse Heart.

Histamine is metabolized by several enzymes in vitro and in vivo. The relevance of this metabolism in the mammalian heart in vivo is unclear. However, histamine can exert positive inotropic effects (PIE) and positive chronotropic effects (PCE) in humans via H2-histamine receptors. In transgenic mice (H2-TG) that overexpress the human H2 receptor in cardiomyocytes but not in wild-type littermate mice (WT), histamine induced PIE and PCE in isolated left or right atrial preparations. These H2-TG were used to investigate the putative relevance of histamine degrading enzymes in the mammalian heart. Histidine, the precursor of histamine, increased force of contraction (FOC) in human atrial preparations. Moreover, histamine increased the phosphorylation state of phospholamban in human atrium. Here, we could detect histidine decarboxylase (HDC) and histamine itself in cardiomyocytes of mouse hearts. Moreover, our data indicate that histamine is subject to degradation in the mammalian heart. Inhibition of the histamine metabolizing enzymes diamine oxidase (DAO) and monoamine oxidase (MAO) shifted the concentration response curves for the PIE in H2-TG atria to the left. Moreover, activity of histamine metabolizing enzymes was present in mouse cardiac samples as well as in human atrial samples. Thus, drugs used for other indication (e.g. antidepressants) can alter histamine levels in the heart. Our results deepen our understanding of the physiological role of histamine in the mouse and human heart. Our findings might be clinically relevant because we show enzyme targets for drugs to modify the beating rate and force of the human heart.