The ins and outs of cyclic di-GMP signaling in Vibrio cholerae

Abstract

The second messenger nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP) governs many cellular processes in the facultative human pathogen Vibrio cholerae. This organism copes with changing environmental conditions in aquatic environments and during transitions to and from human hosts. Modulation of c-di-GMP allows V. cholerae to shift between motile and sessile stages of life, thus allowing adaptation to stressors and environmental conditions during its transmission cycle. The V. cholerae genome encodes a large set of proteins predicted to degrade and produce c-di-GMP. A subset of these enzymes has been demonstrated to control cellular processes – particularly motility, biofilm formation, and virulence – through transcriptional, post-transcriptional, and translational mechanisms. Recent studies have identified and characterized enzymes that modulate or sense c-di-GMP levels and have lead towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae.

Graphical abstract

Introduction

Bacteria use a range of signal transduction systems to sense environmental conditions and launch adaptive responses, enabling them to enhance survival and proliferation by conserving energy. The signaling nucleotide bis (3’-5’) cyclic dimeric guanosine monophosphate (c-di-GMP) has been identified in all major bacterial phyla, and it functions as a second messenger in signal transduction pathways. C-di-GMP is produced by diguanylate cyclases (DGCs), which have GGDEF domains, and degraded by phosphodiesterases (PDEs), which have EAL or HD-GYP domains (Fig 1). The abundance of c-di-GMP metabolizing enzymes, particularly in organisms with complex life cycles, suggests that different c-di-GMP regulatory circuits are activated to regulate cellular processes in response to multiple external cues. The facultative human pathogen Vibrio cholerae, the causative agent of the diarrheal disease cholera, has a complex life cycle involving transitions between aquatic environments and human hosts. The V. cholerae genome boasts a high number of predicted DGC and PDE genes, indicating that c-di-GMP may be regulated in response to a variety of fluctuating environmental parameters during its transmission cycle [1]. In this organism, c-di-GMP promotes motile to sessile transition by repressing motility and enhancing biofilm matrix production. Additionally, c-di-GMP represses V. cholerae virulence. Below, we outline current knowledge and recent advances regarding the mechanisms by which c-di-GMP participates in regulation of these cellular processes, as well as how external cues are sensed and incorporated into c-di-GMP regulatory circuits.

Fig 1.

Model of c-di-GMP signaling. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) harboring catalytic GGDEF domains and degraded by phosphodiesterases (PDEs), which can have EAL or HD-GYP catalytic domains. Bacteria often possess multiple DGCs and PDEs; furthermore, many DGCs and PDEs are multi-domain proteins harboring sensing domains, indicating that their activity may be regulated by environmental signals. The oligoribonuclease Orn regulates the abundance of pGpG. Elevated levels of pGpG are responsible for product inhibition of EAL containing enzymes in Pseudomonas aeruginosa ([60,61]). The V. cholerae genome is encodes 31 GGDEF domain, 12 EAL domain, 10 GGDEF-EAL domain, and 9 HD-GYP domain. Cellular c-di-GMP molecules are sensed by multiple types of receptors that regulate downstream cellular processes at transcriptional, post-transcriptional, and post-translational levels.

C-di-GMP is a key repressor of motility

While the precise molecular mechanisms by which c-di-GMP affects motility in V. cholerae are unclear; high c-di-GMP levels inhibit the production and function of V. cholerae's single polar flagellum [2,3]. Systematic analysis of the impact of c-di-GMP-generating and -degrading enzymes on motility using soft agar motility plates (LB 0.3% agar) at 30°C revealed that the absence of the DGCs CdgD (VCA0697), CdgH (VC1067), CdgK (VC1104), or CdgL (VC2285) enhances motility [4]. Additionally, loss of VpvC (VC2454) leads to increased motility in strains with high c-di-GMP levels [5]. In contrast, lack of predicted PDEs RocS (VC0653) or CdgJ (VC0137) represses motility [4,6]. Lack of some of the c-di-GMP metabolizing enzymes impacts motility phenotype without impacting flagellum production, suggesting that c-di-GMP affects the function of the polar flagellum [4].

Cellular c-di-GMP levels vary in different V. cholerae isolates [7]. Thus, when tested in different V. cholerae genetic backgrounds, additional DGCs and PDEs were shown to govern motility. For example, in the classical biotype, the DGC AcgB (VC1593) and the PDE AcgA (VC1592) regulate motility in low phosphate conditions [8]. The PDE VieA (VC1652) enhances motility in the classical biotype, but not in El Tor [9].

C-di-GMP receptor proteins with PilZ domains play roles in controlling motility. The PilZ domain was the first c-di-GMP binding domain to be characterized, and it has a widespread distribution among bacteria, similar to distributions of GGDEF and EAL domains [10]. The PilZ domain has two consensus motifs, RxxxR and D/NxSxxG, required for c-di-GMP binding [11], and it is found in several proteins affecting flagellar motility [12–15]. The V. cholerae genome encodes five proteins with predicted PilZ domains: PlzA (VC0697), PlzB (VC1885), PlzC (VC2344), PlzD (VCA0042), and PlzE (VCA0735). All of these Plz proteins except PlzB have the two consensus motifs involved in PilZ c-di-GMP binding. PlzC, PlzD, and PlzE were demonstrated to bind c-di-GMP in vitro [16,17]. Furthermore, a structural study revealed that PlzD is found as a dimer in the absence of c-di-GMP, and conformational change occurs upon c-di-GMP binding (Fig 2) [18]. The biological consequences of such conformational changes remain unknown. Analyses of strains lacking Plz proteins have revealed moderate motility phenotypes; PlzB and PlzC serve as enhancers of motility, while PlzD is a repressor (Fig 3A) [4,16]. The molecular basis of motility regulation by Plz proteins in V. cholerae is not yet understood.

Fig. 2.

Structure of c-di-GMP receptors. (A) PilZ domain-containing protein PlzD. Constitutive dimerization of PlzD is mediated by its YcgR-N* domain (green), which adopts the same fold as its PilZ domain (purple). Binding of c-di-GMP to a site involving the linker and the PilZ domain induces an intramolecular conformational change. PDB codes: 1yln [Zhang et al., unpublished]; 2rde [18]. (B) Transcription factor VpsT. The dinucleotide stabilizes a dimer between two VpsT molecules. The dimerization interface is characterized by a signature helix that complements the fold of this subfamily of receiver domains (pink). PDB codes: 3kln and 3klo [22]. (C) MshEN domain-containing proteins. MshE-type ATPases contain the conserved the MshEN domain that binds c-di-GMP through an extended sequence motif in its MshEN_N domain (purple). PDB code: 5htl [29]. (D) RNA aptamers. A class of c-di-GMP-binding riboswitches has been identified as a downstream target of the dinucleotide. Helices P1, P2, and P3 are colored in shades of blue; nucleotides that interact with the nucleotide directly are shown in pink. PDB code: 3irw [52].

Fig. 3.

C-di-GMP signaling modules. (A) The DGCs CdgD, CdgH, CdgL, and CdgK, and the PDEs RocS and CdgJ, regulate a pool of c-di-GMP that contributes to V. cholerae motility. This c-di-GMP is then bound by receptors that regulate motility. Evidence suggests that the PilZ proteins PlzB, PlzC, PlzD, and PlzE bind c-di-GMP and may interact with flagellar motor proteins to control motility. The transcription factor FlrA binds c-di-GMP and controls expression of the flagellar hierarchy.

(B) The PDEs RocS and CdgJ, along with an uncharacterized DGC(s), regulate a pool of c-di-GMP that controls V. cholerae surface attachment. C-di-GMP is bound by the receptor protein MshE, which forms a hexameric ring and controls extension of the MshA pilus. Upon binding c-di-GMP, MshE triggers assembly of the MshA pilus in a process involving the MSHA biogenesis proteins MshG, MshL, and the major pilin MshA (shown in blue).

(C) The DGCs VpvC, CdgA, CdgH, CdgL, CdgK, and VCA0930, and the PDEs RocS, MbaA, CdgC, CdpA, VC1851, and CdgJ regulate c-di-GMP levels and regulate V. cholerae biofilm formation. C-di-GMP is bound by the transcription factors VpsT and VpsR. C-di-GMP binding stabilizes VpsT dimers and allows for enhanced expression of biofilm genes. The function of VpsR binding to c-di-GMP remains unclear.

(D) DGCs and/or PDEs regulate levels of c-di-GMP bound by the riboswitches Vc1 and Vc2. Upon c-di-GMP binding, Vc1 allows translation of GbpA, a critical virulence factor. GbpA facilitates binding to surfaces with GlcNAc, such as intestinal mucus and chitin. Vc2 regulates translation of TfoY, which then activates the type VI secretion system (T6SS). It is not yet clear whether c-di-GMP allows or prevents translation of tfoY.

(E) The PDE VieA, along with an uncharacterized DGC(s), regulates virulence by degrading c-di-GMP, which then reduces production of the virulence regulator ToxT. ToxT regulates expression of the genes required to produce cholera toxin (CT).

At the transcriptional level, c-di-GMP represses transcription of flagellar genes. A large set of proteins is required for a functional flagellum, and their expression is regulated by a well-characterized four level transcriptional hierarchy [19]. Transcriptional activation begins with the alternative sigma factor σ⁵⁴-dependent regulator FlrA (VC2137). C-di-GMP binds to FlrA and alters its activity, impairing FlrA's ability to activate the expression of the flrBC operon [2]. FlrA is an orthologue of FleQ, the master regulator of flagellar gene expression in Pseudomonas aeruginosa [20]. Structural studies showed that FleQ binds to an intercalated dimer of c-di-GMP and identified three conserved sequence motifs (R-switch (LFR¹⁴⁴S), post-Walker A (R¹⁸⁵, N¹⁸⁶) and the ExxxR³³⁴) that contribute to c-di-GMP binding, and all of which are conserved in FlrA [21]. Binding of c-di-GMP to FleQ imposes a change in its oligomeric conformation and acts as an allosteric inhibitor of its ATPase activity. It is yet to be determined if FlrA undergoes similar conformational changes when bound to c-di-GMP. In addition to FlrA, overproduction of the c-di-GMP-dependent transcriptional activator VpsT represses expression of flagellar genes, however the molecular details of this regulation are still being explored [22].

C-di-GMP governs motile-to-sessile life transition and biofilm matrix production

A biofilm is a surface-attached community of bacteria surrounded in a self-secreted protective matrix composed primarily of polysaccharides and proteins. Biofilm formation is critical for the survival and infection cycle of V. cholerae. This process begins with attachment of cells to surfaces, followed by cell division and production of biofilm matrix components. Major components of the V. cholerae biofilm are Vibrio polysaccharide (VPS), which is produced by proteins encoded in the vps-I and vps-II gene clusters, as well as matrix proteins (RbmA, RbmC, and Bap1) that facilitate cell-cell and cell-surface interactions and contribute to biofilm architecture [23–25]. C-di-GMP regulates both initial attachment and matrix production.

Systematic analysis of biofilm phenotypes and vps gene expression of V. cholerae isogenic mutants with in-frame deletions of predicted DGCs or PDEs found that the DGCs CdgA (VCA0074), CdgH, CdgK, CdgL, CdgM (VC1376), and VpvC and the PDEs RocS, CdpA (VC0130), CdgJ, MbaA (VC0703), CdgC (VCA0785), and VC1851 regulate biofilm formation [5,26]. Additionally, when supplied on a multicopy-number plasmid, VCA0939 induces biofilm formation in a quorum sensing-dependent manner [27]. The PDE VieA represses biofilm formation in the classical biotype, but not El Tor [9].

Biofilm formation is initiated by surface attachment, which is controlled by the MshA pilus. This pilus is assembled via polymerization of MshA subunits, and this assembly requires MshE (VC0405), an AAA+ ATPase that extends the pilus using energy from ATP hydrolysis (Fig 3B). A recent study showed that polymerization of MshA subunits is promoted by c-di-GMP binding via the ATPase MshE [17,28]. MshE binds c-di-GMP in its N-terminal domain [18,29]. Furthermore, structure of the MshE N-terminal domain revealed that c-di-GMP binds to the unique binding motifs consisting of a tandem array of two highly conserved 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ [29]. Binding to c-di-GMP activates MshE to polymerize the MshA pili, which facilitates attachment. Loss of mshE or mshA abolishes initial surface attachment, indicating that c-di-GMP-mediated synthesis of MshA pili is a critical early step in biofilm formation [29].

Biofilm matrix production is regulated by c-di-GMP at the transcriptional level by two key transcriptional activators, VpsR (VC0665) and VpsT (VCA0952) [30] (Fig 3C). VpsR directly activates the expression of VPS biosynthesis genes and genes encoding matrix proteins [31]. The domain architecture of VpsR is similar to members of the Enhancer Binding Protein (EBPs) family. It harbors an atypical REC domain at the N-terminus, a central AAA+ domain, and a Fis-like Helix Turn Helix DNA binding domain at the C-terminus. It has been shown through filter-binding assays that VpsR binds c-di-GMP in vitro, with a dissociation coefficient of 1.6 μM [32]. Even though c-di-GMP was shown to be dispensable for DNA binding by VpsR in vitro [32], it is possible that coordination of this second messenger plays a role in the interaction of VpsR with the RNA polymerase or induces conformational changes that promote transcription activation. While VpsR shares sequence similarity with FleQ and FlrA, the AAA+ central domain of VpsR lacks the σ⁵⁴ binding motif (GAFTGA domain) as well as the conserved residues present in FleQ/FlrA orthologues that form the c-di-GMP binding pocket [21]. This would suggest that the mechanism of c-di-GMP binding, and perhaps the conformational changes imposed by binding of c-di-GMP, are different in VpsR-like proteins.

While VpsR is required for expression of biofilm genes, VpsT plays an accessory role; loss of vpsT results in decreased expression of vps genes and matrix protein genes [31,33–35]. It has been demonstrated that a dimer of VpsT binds to intercalated c-di-GMP dimer with an affinity of 3.2 μM [22]. Structural studies revealed that VpsT has an atypical REC domain with an additional α helix (α6) compared to canonical REC domains with a (α/β)₅-fold [22]. The α6 is part of the c-di-GMP dependent dimerization interface, where four amino acid residues with the consensus sequence W[F/L/M][T/S]R, form the nucleotide binding pocket. C-di-GMP binding stabilizes the VpsT dimer conformation (Fig 2). Interestingly, VpsT can undergo c-di-GMP-independent dimerization as well. On the basis of genetic and biochemical studies, it has been proposed that this dimerization interface negatively regulates VpsT activity in the absence of c-di-GMP [22].

C-di-GMP regulates the type VI secretion system

Another V. cholerae cellular process that involves c-di-GMP signaling is the type VI secretion system (T6SS). The T6SS is found in many Gram negative bacteria; it is a contractile structure that injects toxic effector proteins upon contact with neighboring prokaryotic or eukaryotic cells [36–38]. C-di-GMP is involved in a T6SS regulatory network via one of V. cholerae's two c-di-GMP riboswitches.

Riboswitches are cis-acting elements in the 5’ UTR of some mRNA transcripts. In response to a signal, structural rearrangement of riboswitches controls translation or transcriptional termination of a gene product [39,40]. Two types of c-di-GMP riboswitches have been identified in bacteria, class I and class II [41,42]. Class I riboswitches contain a GEMM motif, which is often found in the 5’ UTR of bacterial genes with roles in environment, membrane, or motility functions, including many c-di-GMP signaling genes [41]. V. cholerae encodes two class I riboswitches, Vc1 and Vc2.

Vc1 is found in the 5’ UTR of the gbpA (VCA0811) transcript, which encodes the GlcNAc-binding protein GbpA, a key factor in attachment to surfaces containing N-acetyl-D-glucosamine (GlcNAc) such as intestinal mucus and chitin [41,43]. C-di-GMP activates production of GbpA via binding to Vc1 [44] (Fig 3D).

Vc2 binds c-di-GMP with an affinity of 1 nM and lies upstream of tfoY (VC1722) a master regulator of the T6SS. Studies performed in E. coli, designed to evaluate activity of Vc2 revealed that it can function as an on-switch [41]. Interestingly, a recent report showed increased abundance of TfoY in V. cholerae when levels of c-di-GMP are low [45]. The sensitivity of Vc2 to concentrations of c-di-GMP naturally found in V. cholerae cells and the mechanism by which Vc2 functions remains to be fully unveiled.

C-di-GMP represses virulence

C-di-GMP represses virulence in V. cholerae. The VieSAB three-component system, which regulates c-di-GMP concentration through the phosphodiesterase VieA, is critical for V. cholerae virulence. The kinase VieS phosphorylates VieA's REC domain, activating VieA's PDE activity and instigating degradation of c-di-GMP [46]. VieB acts as an inhibitor of VieS and prevents activation of VieA [47]. Loss of the VieSAB system prevents full expression of the gene encoding key virulence regulator ToxT, as well as the genes encoding cholera toxin (CT) [48]. Contact with mammalian epithelial cells promotes expression of vieA, and the EAL domain of VieA was shown to be involved in depleting c-di-GMP levels and inducing virulence gene expression upon sensing host cell contact [49].

It is important to note that this regulation is strain-specific. In the classical V. cholerae biotype, high c-di-GMP levels lower the amount of ToxT, which in turn lowers virulence gene expression (Fig 3E). El Tor ToxT levels are not altered by high c-di-GMP in vitro; however, there is recent evidence that ToxT does respond to artificially high c-di-GMP in vivo in El Tor strains [32,50]. Hence, the relationship between c-di-GMP and virulence in the El Tor strains remains unclear.

Analysis of gene expression during late stage V. cholerae infection revealed that expression of several DGC and PDE genes are induced during late stage infection. These genes encode the DGCs VC2697, VC1593, and VC2370, and the PDE CdpA [51]. While deletion of these genes individually did not significantly impact colonization or transmission phenotypes, a ΔVC2697 ΔVC1593 ΔVC2370 triple mutant had a 2-3-fold survival defect in stool and pond environments, suggesting that they play a role in transition from host to environment [51] (Fig 3F).

Environmental Signals and Regulation of DGCs and PDEs

Many V. cholerae DGCs/PDEs have sensory domains, suggesting that their activity is controlled by external cues, allowing for c-di-GMP levels to be modulated by environmental parameters. Below, we highlight some of the environmental signals that are known impact cellular c-di-GMP levels (Fig 4).

Fig. 4.

Model for signals modulating c-di-GMP levels. (A) In the aquatic environment, V. cholerae experiences shifts in temperature and likely in oxygen availability. High temperatures (30°C) repress c-di-GMP levels through six DGCs; CdgA, CdgL, CdgH, CdgM, CdgK, and VpvC. Likewise, low temperature (15°C) enhances c-di-GMP production through the same six DGCs. High oxygen levels, such as those expected in the outer edges of mature biofilms, contribute to formation of ferric iron, which can be bound by Vc Bhr-DGC. Upon binding two molecules of ferric iron, Vc Bhr-DGC's activity is repressed. Low oxygen levels, such as those expected deeper inside of a mature biofilm, contribute to formation of ferrous iron. Vc Bhr-DGC activity is enhanced when bound to two molecules of ferrous iron, supporting a model in which V. cholerae biofilm production is enhanced in anoxic environments.

(B) In the intestinal lumen, V. cholerae is exposed to bile acids, which repress expression of one PDE, VC1295, and activity of three DGCs; CdgH, CdgM, and VC1372. This regulation results in an overall increase in c-di-GMP level. Bicarbonate quenches the effect of bile acids on c-di-GMP, suggesting that c-di-GMP levels decrease near intestinal epithelial cells, where bicarbonate levels are higher. Polyamines are present in the human gastrointestinal tract. Norspermidine is bound by the periplasmic sensor protein NspS, which then enhances the PDE activity of MbaA. Spermidine triggers the opposite response, suggesting that it may compete with norspermidine for the NspS binding site. In classical V. cholerae strains, contact with host cells enhances expression of vieA, which leads to production of the PDE and critical virulence factor VieA.

V. cholerae must cope with significant shifts in temperature, both during its residence in aquatic habitats and during transitions between mammalian hosts and the external environment. A recent study showed that low growth temperature enhances c-di-GMP concentration and biofilm formation in V. cholerae [52]. Intracellular c-di-GMP concentration was highest in cells grown at 15°C, followed by 25°C and then 37°C. This temperature-dependent regulation is facilitated by the additive contributions of six diguanylate cyclases: CdgA, CdgH, CdgK, CdgL, CdgM, and VpvC. Temperature was shown to regulate expression of five of the six DGC genes; however, this transcriptional regulation only partially accounts for the increase in c-di-GMP after shifting growth temperature from 37°C to 15°C, indicating that additional post-transcriptional regulation also contributes to increased c-di-GMP at high temperatures.

In the intestinal lumen, V. cholerae is exposed to bile acids which leads to an increase in cellular c-di-GMP concentration [53,54]. The effect of bile acids on c-di-GMP is mediated by three DGCs, CdgH, VC1372, and CdgM, and one PDE, VC1295 [54]. Bile acids regulate the DGCs at the post-transcriptional level and the PDE at the transcription level through unknown mechanisms. Two of the DGCs, CdgH and CdgM, possess sensory domains that may be involved in sensing bile acids. Interestingly, CdgH and CdgM are also involved in the response to temperature, indicating that these proteins are part of multiple c-di-GMP signaling pathways and may be capable of sensing multiple signals [52]. The effect of bile acids on c-di-GMP is quenched by bicarbonate, which causes a local increase in pH [54]. These findings indicate a scenario in which V. cholerae c-di-GMP concentration is finely-tuned and dependent on the cells’ precise location within the host intestine.

Polyamines, which are found within the human gastrointestinal tract, represent another signal modulating cellular c-di-GMP concentration in V. cholerae. The sensor protein NspS regulates activity of a PDE in response to polyamines. Two structurally-similar polyamines, norspermidine and spermidine, regulate V. cholerae c-di-GMP and biofilm formation [55]. Exogenous norspermidine activates V. cholerae biofilm formation through a pathway involving the membrane-bound PDE MbaA and its periplasmic partner protein NspS, which are encoded in the same operon [56]. Evidence suggests that NspS binds norspermidine and then interacts with the periplasmic domain of MbaA and inhibits its PDE activity, thus preventing repression of biofilm genes. Conversely, exogenous spermidine was shown to repress V. cholerae biofilm formation; this repression may occur via competition with norspermidine for the NspS binding site [57,58].

In addition to signals modulating cellular c-di-GMP levels, recent studies revealed mechanisms of activation of DGCs via environmental signals. The V. cholerae enzyme Vc Bhr-DGC (VC1216) possesses a Bhr domain, which can bind two non-heme di-iron atoms in either ferrous or ferric form [59]. Binding to ferrous di-iron enhances the DGC activity of the enzyme, supporting a model in which Vc Bhr-DGC stimulates c-di-GMP concentration and biofilm formation in anoxic environments.

Conclusion

C-di-GMP-mediated phenotypes are critical for the life cycles of many bacteria, including the human pathogen V. cholerae. Therefore, understanding the inputs that influence c-di-GMP production and degradation and the consequences of c-di-GMP signaling is crucial. Significant gains in our knowledge of the regulation of c-di-GMP and the role of c-di-GMP-mediated phenotypes in V. cholerae have been made. We now understand mechanisms of action of DGCs and PDEs and key transcriptional regulators in c-di-GMP signaling, as well as the impact of many environmental cues on c-di-GMP signaling. Due to versatility of the c-di-GMP structure, c-di-GMP-binding proteins employ a wide variety of binding motifs and mechanisms of action, making them difficult to predict computationally. While novel screening methods have facilitated identification of new receptors, the roles of these proteins in V. cholerae biology are yet to be determined [17]. Further studies are needed to reveal the mechanisms of action of known c-di-GMP binding proteins, such as the Plz proteins, as well as to determine if any c-di-GMP effectors remain to be discovered in V. cholerae. Additionally, it is not yet known how c-di-GMP regulates virulence factors, or how c-di-GMP's regulation of biofilm and motility contribute to infection. Further characterization of environmental signals, receptors, and specificity in c-di-GMP signaling will greatly inform our understanding of how bacteria adapt to environmental stressors, and may allow for development of novel strategies to cope with problems posed by biofilm, such as biofouling and antibiotic resistance.

Highlights.

• A plethora of c-di-GMP metabolizing enzymes impact diverse cellular processes in Vibrio cholerae.

• C-di-GMP receptors are diverse and target processes are controlled by different mechanisms.

• Variety of external signals are integrated into c-di-GMP signaling circuitry.

• Mechanisms and target processes of new c-di-GMP receptors are yet to be determined.