Progress in understanding of the molecular basis underlying functional diversification of cyclic di-nucleotide turnover proteins

Cyclic di-GMP was the first cyclic di-nucleotide second messenger described, presaging the discovery of additional cyclic di-nucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, to include both enzymatically active and inactive members with a subset involved in synthesis and degradation of other cyclic di-nucleotides. Here we summarize current knowledge of sequence and structural varitions that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL and HD-GYP domain proteins.

Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p) ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Delta rel(Msm) and Delta dcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p) ppGpp and c-di-GMP in mycobacteria. We have shown that both (p) ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Delta rel(Msm) and Delta dcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p) ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p) ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p) ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Delta rel(Msm) and Delta dcpA strains are defective in biofilm formation. In this work, the overproduction of (p) ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p) ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p) ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p) ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (Delta rel(Msm)) or c-di-GMP synthesis (Delta dcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Delta rel(Msm) and Delta dcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Synthesis and Characterization of a Fluorescent Analogue of Cyclic di-GMP

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

UNLABELLED: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

IMPORTANCE: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

A SUBSET OF 3´ TO 5´ EXORIBONUCLEASES ARE THE PRIMARY ENZYMES RESPONSIBLE FOR THE DEGRADATION OF PGPG IN CYCLIC DI-GMP SIGNALING

Bis-(3´-5´)-cyclic dimeric guanosine monophosphate, or cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates processes such biofilm formation, motility, and virulence. C-di-GMP is synthesized by diguanylate cyclases (DGCs), while phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMPs by previously unidentified enzymes termed PDE-Bs. To identify the PDE-B responsible for pGpG turnover, a screen for pGpG binding proteins in a Vibrio cholerae open reading frame library was conducted to identify potential pGpG binding proteins. This screen led to identification of oligoribonuclease (Orn). Purified Orn binds to pGpG and can cleave pGpG to GMP in vitro. A deletion mutant of orn in Pseudomonas aeruginosa was highly defective in pGpG turnover and accumulated pGpG. Deletion of orn also resulted in accumulation c-di-GMP, likely through pGpG-mediated inhibition of the PDE-As, causing an increase in c-di-GMP-governed auto-aggregation and biofilm. Thus, we found that Orn serves as the primary PDE-B enzyme in P. aeruginosa that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. However, not all bacteria that utilize c-di-GMP signaling also have an ortholog of orn, suggesting that other PDE-Bs must be present. Therefore, we asked whether RNases that cleave small oligoribonucleotides in other species could also act as PDE-Bs. NrnA, NrnB, and NrnC can rapidly degrade pGpG to GMP. Furthermore, they can reduce the elevated aggregation and biofilm formation in P. aeruginosa ∆orn. Together, these results indicate that rather than having a single dedicated PDE-B, different bacteria utilize distinct RNases to cleave pGpG and complete c-di-GMP signaling. The ∆orn strain also has a growth defect, indicating changes in other regulatory processes that could be due to pGpG accumulation, c-di-GMP accumulation, or another effect due to loss of Orn. We sought to investigate the genetic pathways responsible for these growth defect phenotypes by use of a transposon suppressor screen, and also investigated transcriptional changes using RNA-Seq. This work identifies that c-di-GMP degradation intersects with RNA degradation at the point of the Orn and the functionally related RNases.

Backbone-modified amphiphilic cyclic di- and tetrasaccharides

Synthesis of amphiphilic, cyclic di- and tetrasaccharides, which incorporate a methylene moiety at the inter-glycosidic bond, is reported. The amphiphilic properties of the new cyclic tetrasaccharide host were identified through assessing the solubilities of guests in aqueous and in organic solvents. The glycosidic bond stability of the cyclic tetrasaccharide under aqueous acidic condition was also verified.

IR/Raman spectroscopy and DFT calculations of cyclic di-amino acid peptides. Part III: comparison of solid state and solution structures of cyclo(L-Ser-L-Ser)

B3-LYP/cc-pVDZ calculations of the gas-phase structure and vibrational spectra of the isolated molecule cyclo(L-Ser-L-Ser), a cyclic di-amino acid peptide (CDAP), were carried out by assuming C-2 symmetry. It is predicted that the minimum-energy structure is a boat conformation for the diketopiperazine (DKP) ring with both L-Beryl side chains being folded slightly above the ring. An additional structure of higher energy (15.16 kJ mol(-1)) has been calculated for a DKP ring with a planar geometry, although in this case two fundamental vibrations have been calculated with imaginary wavenumbers. The reported X-ray crystallographic structure of cyclo(L-Ser-L-Ser), shows that the DKP ring displays a near-planar conformation, with both the two L-Beryl side chains being folded above the ring. It is hypothesized that the crystal packing forces constrain the DKP ring in a planar conformation and it is probable that the lower energy boat conformation may prevail in the aqueous environment. Raman scattering and Fourier-transform infrared (FT-IR) spectra of solid state and aqueous solution samples of cyclo(L-Ser-L-Ser) are reported and discussed. Vibrational band assignments have been made on the basis of comparisons with the calculated vibrational spectra and band wavenumber shifts upon deuteration of labile protons. The experimental Raman and IR results for solid-state samples show characteristic amide I vibrations which are split (Raman:1661 and 1687 cm(-1), IR:1666 and 1680 cm(-1)), possibly due to interactions between molecules in a crystallographic unit cell. The cis amide I band is differentiated by its deuterium shift of ~ 30 cm(-1), which is larger than that previously reported for trans amide I deuterium shifts. A cis amide II mode has been assigned to a Raman band located at 1520 cm(-1). The occurrence of this cis amide II mode at a wavenumber above 1500 cm(-1) concurs with results of previously examined CDAP molecules with low molecular weight substituents on the C-alpha atoms, and is also indicative of a relatively unstrained DKP ring.

Discrete cyclic di-GMP-dependent control of bacterial predation versus axenic growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

GABA, serotonin, muscarinic receptors - their second messengers and transcription factors functional regulation in the brain regions of hypoxic neonatal rats: Resuscitation with glucose, oxygen and epinephrine

The present study was designed to investigate the protective effect of glucose, oxygen and epinephrine resuscitation on impairment in the functional role of GABAergic, serotonergic, muscarinic receptors, PLC, BAX, SOD, CAT and GPx expression in the brain regions of hypoxia induced neonatal rats. Also, the role of hormones - Triiodothyronine (T3) and insulin, second messengers – cAMP, cGMP and IP3 and transcription factors – HIF and CREB in the regulation of neonatal hypoxia and its resuscitation methods were studied. Behavioural studies were conducted to evaluate the motor function and cognitive deficit in one month old control and experimental rats. The efficient and timely supplementation of glucose plays a crucial role in correcting the molecular changes due to hypoxia, oxygen and epinephrine. The sequence of glucose, epinephrine and oxygen administration at the molecular level is an important aspect of the study. The additive neuronal damage effect due to oxygen and epinephrine treatment is another important observation. The corrective measures by initial supply of glucose to hypoxic neonatal rats showed from the molecular study when brought to practice will lead to healthy intellectual capacity during the later developmental stages, which has immense clinical significance in neonatal care.

Analysis of second messengers in peripheral and central circadian pacemakers

In früheren Experimenten konnte gezeigt werden, dass die sekundären Botenstoffe cAMP, cGMP und IP3 in der olfaktorischen Signaltransduktionskaskade bei Manduca sexta involviert sind. Während cGMP Perfusionen in die Antenne die Pheromonwahrnehmung tageszeitabhängig adaptierten, führten cAMP Perfusionen zu einer tageszeitabhängigen Sensitisierung, ähnlicher der von Octopamin (OA). Daher wurde hypothetisiert, dass eine tageszeitabhängige Oszillation antennaler OA Level sowie der intrazelluläre Kalziumkonzentration in einer Schwankung von sekundären Botenstoffen resultieren könnte. Diese Hypothese wurde mittels biochemischen Nachweißverfahren in der Antenne von M. sexta und Rhyparobia maderae überprüft. Tatsächlich konnten in der Antenne des Tabakschwärmers tageszeitabhängige Unterschiede in der OA-, cAMP- und IP3-, aber nicht in der cGMP Konzentration, nachgewiesen werden. Während die cAMP- und OA Oszillationen einander sehr ähnelten und die Maxima in der Paarungsphase aufzeigten, korrelierte der IP3 Verlauf sehr stark mit dem Flug- bzw. Fressverhalten. Diese Korrelationen konnten auch in der Madeira Schabe beobachtet werden, in der darüber hinaus gezeigt werden konnte, dass antennale cAMP- und IP3 Level von dem circadianen Uhrwerk gesteuert werden. Zudem wurde herausgefunden, dass OA die cAMP- und teilweise auch die IP3- Spiegel reguliert. Demgegenüber beeinflusste Kalzium die Konzentration aller untersuchten sekundären Botenstoffe. Daher wird angenommen, dass die intrazelluläre Kalziumkonzentration aber auch der antennale OA Level kritische Faktoren bei der Regelung der olfaktorischen Sensitivität sind. Da Oszillationen von sekundären Botenstoffen in mutmaßlichen, peripheren Schrittmacher nachgewiesen wurden, wurde untersucht, ob sie auch im circadianen Schrittmacher der Madeira Schabe oszillieren und ob das Neuropeptid pigment-dispersing factor (PDF), ein entscheidender Kopplungsfaktor des Uhrwerks in Insekten, diese Rhythmen generieren könnte. Es konnte gezeigt werden, dass PDF die cAMP Synthese steigert. Darüber hinaus wurden bimodale cAMP Oszillationen unter licht-dunkel Bedingungen beobachtet, welche unter konstanten Umweltbedingungen verblieben. Daher wird angenommen, dass PDF Freisetzung zelluläres cAMP erhöht über das das circadiane Uhrwerk synchronisiert wird.

Generation of second messengers in Plasmodium

Signalling in malaria parasites is a field of growing interest as its components may prove to be valuable drug targets, especially when one considers the burden of a disease that is responsible for up to 500 million infections annually. The scope of this review is to discuss external stimuli in the parasite life cycle and the upstream machinery responsible for translating them into intracellular responses, focussing particularly on the calcium signalling pathway. (C) 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur.