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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

Cyclic-di-GMP (c-di-GMP) regulates many important bacterial processes. Freely diffusible intracellular c-di-GMP is determined by the action of metabolizing enzymes that allow integration of numerous input signals. c-di-GMP specifically regulates multiple cellular processes by binding to diverse target molecules. This review highlights important questions in research into the mechanisms of c-di-GMP signalling and its role in bacterial physiology.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

Cyclic-di-GMP (c-di-GMP) is a small, diffusible intracellular molecule that serves as single integrated output from multiple sensory inputs. An important advantage of second messenger systems is that its output is modulated by enzymatic synthesis and degradation, which is more rapid than the transduction of sensory information through gene transcription and translation. Current knowledge indicates that c-di-GMP is the second messenger most diversely utilized by bacteria. Its synthesis and degradation is regulated by many different molecules with various predicted sensing domains, indicating that its cellular level is controlled by diverse extracellular inputs. Several different classes of molecules bind c-di-GMP and, through this binding, modulate a variety of downstream processes, including flagellar motility, adhesion to biotic and abiotic surfaces, the cell cycle, and the synthesis of extracellular carbohydrate polymers including cellulose.

c-di-GMP synthesis and degradation is accomplished by proteins containing clearly defined enzymatic domains (Fig. 1). c-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) that share a conserved domain containing the amino acid motif GGDEF. c-di-GMP is degraded by phosphodiesterases (PDEs) that contain one of two conserved domain families: one defined by the EAL motif and the other by an HD-GYP motif. Many genomes contain multiple paralogues of dgc and pde genes, demonstrating that their products metabolize the same small molecule, c-di-GMP (http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html). In order to exert its effects, c-di-GMP specifically binds to downstream effector molecules. Thus, c-di-GMP network sensors (DGCs and PDEs) sense internal or external signals and translate these signals into c-di-GMP levels, which then modulate the function of c-di-GMP binding molecules, resulting in an alteration of the physiology and behaviour of the cell. This system, though complex in many bacterial cells, allows c-di-GMP to regulate processes at multiple levels, including effects on transcriptional, translational and post-translational functions.

image

Figure 1. c-di-GMP metabolism. c-di-GMP is synthesized from GTP by diguanylate cyclases containing GGDEF domains (green) and is degraded into pGpG by phosphodiesterase domains containing either EAL or HD-GYP domains (blue). Depending on c-di-GMP binding receptor expression patterns and binding affinities to c-di-GMP, different receptors (red) will be activated. Binding receptors can be highly diverse and include proteins with a variety of domains as well as RNA motifs (see text).

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Second-messenger signalling allows a rapid cellular response to external signals

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

The rapid nature of second messenger signalling allows an almost immediate cellular response to changing environmental conditions. Adaptation to changing conditions frequently involves modulation of the activities of protein complexes that control cellular behaviour, such as the flagellar motility apparatus. Such complexes are large macromolecular structures that are energetically costly and time-consuming to build. De novo transcription and translation of these complexes would require precious time and energy when immediate adaptation to the environment is needed. Allosteric control and post-translational regulation by second messenger turnover is both more energetically favourable, and much faster, than regulation by synthesis and degradation of complex constituents (Tuckerman et al., 2009).

The control of cellular motility by c-di-GMP signalling is the best illustration to date of the importance of ac-di-GMP-controlled rapid response to changing environmental conditions. c-di-GMP levels control flagellar motility of Enteric and likely other bacteria as a result of the interaction of a c-di-GMP binding protein with the cytoplasmic components of the flagellum complex (Boehm et al., 2010; Fang and Gomelsky, 2010; Paul et al., 2010). The bacterial flagellum is a large, complex macromolecular structure consisting of a propeller made of flagellum and a rotary motor consisting of rotor and stator proteins. Flagellar rotation is powered by proton flux across inner membrane channels residing in the motor complex. Transformation of chemical energy from proton influx into torque is believed to involve direct electrostatic interactions between stator and rotor proteins. Swimming behaviour is determined by the direction of flagellar rotation: counterclockwise (CCW) rotation favours smooth swimming, whereas clockwise (CW) rotation induces bacterial tumbling and a random change in direction. Many flagellar constituents are structural components that form the flagellum or are involved in its assembly, while others act to regulate the activity of the flagellar motor. In S. Typhimurium and E. coli, one of these flagellar regulator proteins, YcgR, binds c-di-GMP at a relatively high affinity (Christen et al., 2007). c-di-GMP-bound YcgR inhibits flagellar motility by binding to the motor–stator complex and modulating its function (Boehm et al., 2010; Fang and Gomelsky, 2010; Paul et al., 2010). This effect of c-di-GMP-bound YcgR can be visualized by a decrease in swimming on a solid surface for S. Typhimurium and E. coli (Ryjenkov et al., 2006). The molecular mechanisms of this inhibition have been worked out in some detail. In E. coli, c-di-GMP-bound YcgR was shown to bind directly to the stator protein MotA, and was hypothesized to increase electrostatic interactions between MotA and the rotor protein FliG, thereby acting as a brake (Boehm et al., 2010). In another study, c-di-GMP-bound YcgR was found to interact with the flagellar switch-complex proteins FliG and FliM (Paul et al., 2010). This interaction reduces the efficiency of torque generation and induces CCW motor bias due to disruption of the interaction between FliG and MotA. A third group also showed that c-di-GMP-bound YcgR binds FliG and induces a CCW motor bias (Fang and Gomelsky, 2010). A CCW bias might inhibit swimming motility by reducing the ability of cells to change direction when their swimming is unbeneficial.

YcgR-based regulation of flagellar machinery is determined by the c-di-GMP-bound state of YcgR; thus YcgR activity, and therefore bacterial motility, is controlled post-translationally by enzymatic synthesis and degradation of c-di-GMP. One c-di-GMP-metabolizing enzyme that allows this rapid response has been identified. The flagellar regulator enzyme YhjH is a PDE that is required for maintaining c-di-GMP levels that are low enough to prevent YcgR from inhibiting motility, thereby allowing maximum swimming speed (Ko and Park, 2000). It is likely that, upon sensing conditions that favour a decrease in motility, DGCs increase the concentration of c-di-GMP and/or YhjH is inactivated, c-di-GMP then binds to YcgR, which translates the c-di-GMP signal to modulate motility by direct interaction with motor components. This conversion could potentially occur in seconds, since YcgR transitions from the unbound state to the bound state within a matter of seconds when c-di-GMP levels increase to levels greater than its binding constant [kon 8.49e+5 s−1 M−1; koff 2.49e−2 s−1 (Christen et al., 2010)]. DGCs and PDEs that have been enzymatically characterized are capable of metabolizing c-di-GMP on this rapid biological timescale (Christen et al., 2005; 2006). It is likely that other large macromolecular complexes that are controlled by c-di-GMP also demonstrate a beneficial rapid response property.

c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

c-di-GMP modulates many diverse processes in the cell in addition to motility including exopolysaccharide biosynthesis, the synthesis of extracellular adhesins, developmental transitions of dividing cells and bacterial virulence. The ability of a second messenger to have numerous effects on cellular behaviour lies in the diversity of c-di-GMP receptors, which act as effector molecules. These receptors monitor the c-di-GMP level in the cell and translate it to a specific behavioural response.

One method by which c-di-GMP effectors respond to intracellular synthesis or degradation of c-di-GMP is through a c-di-GMP-binding protein domain termed the PilZ domain. Such a domain is present in YcgR, the flagellar brake. The molecular details of how several PilZ-domain proteins bind c-di-GMP as an intercalated dimer are known from crystal and NMR structures (Benach et al., 2007; Ramelot et al., 2007; Ko et al., 2010; Habazettl et al., 2011; Shin et al., 2011). Proteins that make use of PilZ domains to sense c-di-GMP take a variety of forms. Though some PilZ-domain proteins consist solely of a PilZ domain, others contain a variety of additional domains to achieve their unique functionality. YcgR links a PilZ domain to a YcgR-N protein–protein interaction domain: the PilZ domain binds c-di-GMP, causing structural changes in the protein, and then both domains make physical interactions with the flagellar machinery.

In addition to modulating protein–protein interactions, c-di-GMP also regulates many enzymes by binding to a PilZ domain that allosterically affects enzymatic activity. The cellulose synthesis enzyme of Salmonella Typhimurium, BcsA, is one example. This enzyme contains a cytoplasmic PilZ domain, which is thought to regulate the enzymatic activity of a periplasmic cellulose synthesis domain, based on its c-di-GMP-bound state (Zogaj et al., 2001). Alginate production by Pseudomonas aeruginosa is also regulated by binding of c-di-GMP to the PilZ domain of the predicted alginate synthesis enzyme Alg44 (Merighi et al., 2007). PilZ domains have also been identified in proteins that contain GGDEF and EAL domains, helix-turn-helix DNA binding domains, and a variety of protein–protein interaction domains (Amikam and Galperin, 2006), indicating that the inclusion of a PilZ domain is a widespread tactic to respond to cellular c-di-GMP levels. This is exemplified by the fact that the genomes of many bacterial species encode a multitude of PilZ domains. One example of this is the Pseudomonas aeruginosa genome, which encodes eight PilZ domains, most without a currently defined function.

Another mechanism by which effector proteins respond to c-di-GMP levels is through degenerate GGDEF and EAL domains. Proteins that contain GGDEF or EAL domains, which have lost catalytic activity, can bind c-di-GMP at an allosteric c-di-GMP-binding site of a GGDEF domain, or the catalytic site of an EAL domain, respectively. One example of this is the LapD protein from Pseudomonas fluorescens, which contains both GGDEF and EAL domains. The GGDEF and EAL domains are degenerate and show no in vitro enzymatic activity. Instead, LapD is a c-di-GMP receptor, binding c-di-GMP to its degenerate EAL domain (Newell et al., 2009).

Other non-PilZ-, non-GGDEF/EAL-domain proteins have evolved the ability to sense c-di-GMP through the evolution of domains that previously had some other function, such as binding other small nucleic acids. One example of this is Xanthomonas campestris Clp protein, a cyclic-AMP-receptor-protein (Crp) homologue, that is a transcription factor (Chin et al., 2010). While most Crp proteins bind cAMP, Clp binds c-di-GMP. This binding modulates the expression of a number of genes that are involved in Xanthomonas virulence (Chin et al., 2010). In addition to binding protein partners, c-di-GMP has also been shown to specifically bind specialized RNA domains to regulate translation of target mRNAs (Sudarsan et al., 2008). Hundreds of potential mRNAs encoding this domain have been predicted from DNA sequence and in some cases identified by RNA analysis. Many of these c-di-GMP-binding proteins and mRNA domains may remain undiscovered in bacterial genomes.

The diversity of its receptors allows c-di-GMP to control cellular behaviour on a transcriptional level [through binding to transcription factors like X. campestris Clp and P. aeruginosa FleQ (Hickman and Harwood, 2008)], on a translational level (through interactions with mRNA domains or mRNA-processing enzymes (Tuckerman et al., 2011)), and on a post-translational level (by allosteric regulation of enzymatic complexes or other effector molecules such as YcgR). This allows c-di-GMP signalling to rapidly change cellular behaviour on a post-translational level (as mentioned above) and also to regulate the composition of cellular proteins in a more long-term fashion.

Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

The enzymes that participate in c-di-GMP turnover, DGCs and PDEs, are regulated by a variety of environmental conditions. This allows precise signal integration based on cell sampling of the environment and transduction of this information into specific c-di-GMP levels within the cell. Environmental regulation of DGC and PDE activity is most likely accomplished by the diverse sensory and regulatory domains frequently associated with these proteins (Jenal, 2004; Romling and Amikam, 2006; Tamayo et al., 2007; Yan and Chen, 2010). Common sensory domains found in proteins containing GGDEF or EAL include those termed PAS, HAMP and REC domains. These domains have been linked to the sensing of small molecules, redox potential, light, voltage, oxygen, nutrients, osmolarity, antibiotics, homoserine lactones, and a number of other signals. Although several DGCs and PDEs have been identified that respond to these inputs, or that are involved in biological processes associated with specific inputs (Tal et al., 1998; Ryan et al., 2006; Barends et al., 2009; Tuckerman et al., 2009), few actual signals have been conclusively demonstrated to activate these predicted sensing domains. Many bacterial species encode dozens of c-di-GMP-metabolizing enzymes with a variety of predicted sensing domains, allowing the cell to translate many environmental signals into second messenger levels. For example, of the 41 putative c-di-GMP-metabolizing enzymes encoded by Pseudomonas aeruginosa, almost all contain at least one predicted sensory domain (Kulasakara et al., 2006). Because the GGDEF and EAL domains are modular, genetic shuffling can generate new cellular sensors that can interface with the cellular c-di-GMP pool. This genetic expansion may have led to the remarkable diversity of sensing abilities that are observed in DGCs and PDEs, which can also extend to individual strains within specific species.

Since c-di-GMP signalling controls important cellular behaviours, it is likely that the levels of c-di-GMP are tightly regulated in the cell in response to environmental conditions. This is exemplified by the DosC/DosP/PNPase system in E. coli, in which small differences in affinity of the DGC and PDE sensing domains for their ligand could cause a switch between net c-di-GMP production and degradation. In this system, DosC (a DGC) and DosP (a PDE) sense oxygen levels through two different haem-containing sensor domains. DosC binds molecular oxygen via a GCS domain with an affinity of 21 µM (Tuckerman et al., 2009). DosP binds oxygen via a PAS domain with an affinity of 74 µM, which activates its phosphodiesterase activity (Delgado-Nixon et al., 2000; Tuckerman et al., 2009). These enzymes are transcribed together and physically interact with each other (Mendez-Ortiz et al., 2006; Tuckerman et al., 2009). DosC and DosP co-purify in a complex that contains mRNA and the RNA modifying enzyme PNPase, which is a c-di-GMP receptor. The RNA processing activity of this complex is modulated by oxygen (Tuckerman et al., 2011). Thus information on oxygen levels is integrated by DosC and DosP, which translate this information into a c-di-GMP signal, resulting in the regulation of the mRNA processing activity of PNPase. The generation of a complex in which both DGC and PDE activities are controlled by oxygen levels allows rapid activation and deactivation of PNPase. This illustrates the importance to the cell of precise, rapid signal integration in c-di-GMP signalling and might indicate the utility in some cases of having both cyclase and phosphodiesterase domains in the same molecule.

Though they respond to diverse environmental signals, all paralogous DGCs produce the same small, diffusible second messenger. However, many DGCs and PDEs can be linked to different downstream effects. Given the diversity of inputs and outputs in the c-di-GMP signalling network, how is pathway signalling specificity achieved? It is tempting to speculate that DGCs and PDEs generate and maintain local pools of c-di-GMP that target only local effector molecules. However, the small size of the bacterial cell (2 µm3 for E. coli) (Schulz and Jorgensen, 2001) and the rapid diffusion of small molecules in the cell cytoplasm (270–780 µm s−1 for cAMP) (Zaccolo et al., 2006) make it unlikely that this could be achieved in the absence of cellular compartmentalization, as equilibrium of c-di-GMP concentration in the bacterial cell cytoplasm would be reached in milliseconds. One method to achieve signalling specificity might be based on the affinity of c-di-GMP receptors. In this case, receptors would vary in their affinities for c-di-GMP, which would allow differential binding of these receptors to c-di-GMP based on the specific concentration of the second messenger in the cell. In the same vein, the activities of c-di-GMP-metabolizing enzymes could be regulated to generate specific levels of c-di-GMP. For example, many DGCs may possess feedback inhibition, which prevents the production of c-di-GMP above a certain concentration (Christen et al., 2006). It is also possible that differential expression of DGCs and PDEs affects pathway specificity – Sommerfeldt et al. analysed the expression patterns of all 28 GGDEF/EAL genes of E. coli and showed that most of them demonstrated altered expression levels at different growth phases and different temperatures (Sommerfeldt et al., 2009). These mechanisms could allow the transduction of a specific environmental input into a particular cellular response.

Differential distribution of c-di-GMP in the cell cycle and after cell division

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

In addition to responding to external environmental conditions, c-di-GMP levels are also modulated by internal cues such as those that occur during developmental progression or cellular division. After cellular division, several bacterial species have been shown to produce daughter cells that maintain significantly different, asymmetrical levels of c-di-GMP, and thus differ in their cellular behaviours (Christen et al., 2010). This is best illustrated in Caulobacter crescentus, which is a model organism for cell cycle control since the cycle can be easily synchronized in populations of laboratory-adapted cells. In C. crescentus each cellular division results in a mother cell that is amotile and attached to abiotic surfaces via an adhesive structure called the stalk, and a daughter cell that is free-living and flagellated. The stalked cell may continue to divide again, but the flagellated cell will not divide until it attaches to an abiotic surface and differentiates into a new stalked cell. These behaviours are regulated by differential levels of c-di-GMP. Using a c-di-GMP-sensing FRET biosensor constructed from S. Typhimurium YcgR (Kd = 195 nM), Christen et al. showed an asymmetrical distribution of c-di-GMP in C. cresentus daughter cells in which the flagellated cell had levels lower than 100 nM and the stalked cell had levels higher than 500 nM (Christen et al., 2010). High c-di-GMP levels are maintained in the stalked cell by activated PleD (a DGC), which is targeted to the differentiating stalked old cell pole (Paul et al., 2004). Indeed, cells lacking pleD generated daughter cells in which both cells exhibited c-di-GMP levels below the level of detection of the YcgR biosensor, whereas in a pleC mutant, in which activated PleD localizes to both cell poles, c-di-GMP levels were increased in both daughter cells (Christen et al., 2010). These different c-di-GMP levels are translated into the different behaviours observed in daughter cells. The high level of c-di-GMP in the stalked cell allows cellular division, as c-di-GMP binds to the degenerate GGDEF-domain protein PopA (Kd of 2.4 µM), which facilitates the degradation of the important cell cycle inhibitor CtrA, thus permitting cell replication to occur (Quon et al., 1998; Duerig et al., 2009). In the flagellated cell, the level of c-di-GMP is too low to stimulate the degradation of CtrA, so cell replication is repressed (McGrath et al., 2006). Levels of c-di-GMP in the flagellated cell are also speculated to be below the level at which they bind DgrA, a C. cresentus homologue of YcgR, and thus would not inhibit motility (Kd of 50 nM) (Christen et al., 2007). Theoretically, upon entrance into conditions in which a flagellated cell differentiates into a stalked cell, c-di-GMP levels rise, first inhibiting motility by binding to DgrA, then relieving cell cycle repression by binding to PopA.

Why generate daughter cells with different cellular behaviours? Diversifying progeny could have great survival value, as it increases the chances that at least one cell will live to replicate. Generating one motile and one amotile cell could be one common mechanism of diversification. Indeed, Pseudomonas aeruginosa daughter cells also demonstrate asymmetrical distribution of c-di-GMP, though in this case, the mother cell keeps the single polar flagella and maintains low c-di-GMP and is motile, while the daughter cell maintains high c-di-GMP and is amotile (Christen et al., 2010). Thus, a P. aeruginosa mother cell abandons its young to colonize new soils, while in C. cresentus, the mother cell remains anchored and the daughter swims away to probe other environments (Fig. 2). In addition to generating motility differences, asymmetrical distribution in daughter cells probably affects other c-di-GMP-related behaviours, some of which remain to be discovered. Aflagellate species such as Klebsiella also demonstrate asymmetrical distribution (Christen et al., 2010), implying that the benefits of diversifying c-di-GMP-regulated behaviours in daughter cells may extend beyond flagellar motility. This diversity may confer an advantage in variable, rapidly changing environments, in which selective pressure gives the appearance that the bacterial species is hedging its bets by generating progeny that are specialized for survival in different environmental conditions. Generating individuals with diverse behaviours, from genetically identical parents, could thus lead to the preservation of the species.

image

Figure 2. Influence of c-di-GMP on bacterial dispersal in different bacteria. Caulobacter life cycle is intimately linked to temporal changes in second messenger levels. The stalked cell, which is attached to abiotic surfaces, contains high c-di-GMP. c-di-GMP is required for the cell cycle to proceed. Additionally, c-di-GMP may be required for formation of exopolysaccharides in the stalk structure. Cell division produces a flagellated cell with low second messenger level and a stalked cell with high levels. Similarly, in Pseudomonas, cell division produces a progeny in which c-di-GMP is asymmetrically distributed. The cell with high c-di-GMP may produce an exopolysaccharide capsule that may aid in attachment to abiotic surfaces. In both bacteria, c-di-GMP levels are low in flagellated cells, allowing full flagella motility and subsequent dispersal away from the sessile cells. In Caulobacter, the flagellum is synthesized in a newly formed pole whereas in Pseudomonas, the flagellum is retained in the old pole.

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Eukaryotic sensing of c-di-GMP

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

Although c-di-GMP signalling is ubiquitous in bacteria, no predicted synthesis genes are found in Archaebacteria or Eukaryotes. The eukaryotic innate immune system is capable of identifying pathogens by recognizing pathogen-associated molecular patterns derived from the conserved domains such as those found in lipopolysaccharides, peptidoglycan, toxins, toxin delivery systems and flagellin. It is therefore possible that eukaryotes also use c-di-GMP as a marker for bacterial presence due to its ubiquity. Indeed, it has been shown that c-di-GMP has immunostimulatory properties. Two studies showed that pretreatment with c-di-GMP protected mice against subsequent challenge with Klebsiella pneumoniae or Staphlyococcus aureus (Karaolis et al., 2007a,b).

Since microbe-specific signals are recognized by a variety of eukaryotic receptors, it is possible that there exists a eukaryotic sensor for c-di-GMP. Recently, McWhirter et al. showed that transfected c-di-GMP induces production of type 1 interferon in bone marrow macrophages (McWhirter et al., 2009). This response required downstream signalling components that are also required for the type I interferon response to cytosolic DNA and RNA. Both c-di-GMP and nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and co-regulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. STING, an ER protein shown to be required for intracellular DNA-mediated, type I interferon-dependent innate immunity (Ishikawa et al., 2009), is also required for the recognition of c-di-GMP and another bacterial second messenger, c-di-AMP (Sauer et al., 2011). Research over the next few years should define whether bacterial and viral nucleic acids in general trigger this inflammatory response through a common receptor, or whether each unique signal, such as c-di-GMP, has its own individual receptor.

Perspectives

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References

c-di-GMP was first discovered as an allosteric activator of cellulose synthesis in Glucoacetobacter xylinus in the 1980s (Ross et al., 1987). Since then, much knowledge has been accrued regarding this important signalling molecule, though many important unknowns remain. Many bacterial species encode a staggering number of c-di-GMP components in their genomes, implying that c-di-GMP signalling networks in these organisms are quite complex (Galperin et al., 2010). However, in many cases the specific inputs controlling c-di-GMP metabolism, the mechanisms by which c-di-GMP regulates specific cellular behaviours, and the behaviours themselves are not known.

Technical advances in c-di-GMP research have the potential to allow greater understanding of c-di-GMP signalling networks and to provide therapeutics for some important human pathogens. Recently, the design of FRET-based biosensors allowed c-di-GMP levels to be measured in live bacterial single cells (Christen et al., 2010). This should promote real-time monitoring of c-di-GMP, which will elucidate the effects of environmental conditions on c-di-GMP levels in individual cells. The development of chemical inhibitors that target c-di-GMP-metabolizing enzymes, completely inhibiting c-di-GMP synthesis or degradation, will allow better characterization of the effects of this second messenger on cellular behaviour (Kline et al., 2008; Ching et al., 2010). Chemical inhibitors may provide a better way to determine the contribution of c-di-GMP to bacterial physiology than genetic reduction or overexpression manipulations, which can result in secondary effects thereby confounding results. Even if such inhibitors are not lethal to bacteria, dysregulation of c-di-GMP could cause inappropriate pathogen behaviours resulting in alteration of virulence factor production or, particularly in the case of biofilm infections, increased antibiotic efficacy. Thus, c-di-GMP signalling is an important target of research for both a basic understanding of bacterial physiology and for its potential clinical and therapeutic significance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Second-messenger signalling allows a rapid cellular response to external signals
  5. c-di-GMP signalling affects diverse cellular behaviours through a variety of c-di-GMP binding molecules
  6. Sensory domains in DGCs and PDEs integrate environmental signals to alter c-di-GMP levels
  7. Differential distribution of c-di-GMP in the cell cycle and after cell division
  8. Eukaryotic sensing of c-di-GMP
  9. Perspectives
  10. References
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