Critical Review Cyclic Di-nucleotide Signaling Enters the Pauline Schaap* Eukaryote Domain

College of Life Sciences, University of Dundee, Dundee, UK

Abstract

Cyclic (c-di-GMP) is the prevalent intracellular signaling inter- which are either produced endogenously in response to for- mediate in . It triggers a spectrum of responses that eign DNA or by invading bacterial pathogens, trigger the cause bacteria to shift from a swarming motile phase to innate immune system by activating the expression of inter- sessile biofilm formation. However, additional functions for feron genes. STING, the human c-di-NMP receptor, is con- c-di-GMP and roles for related molecules, such as c-di-AMP served throughout metazoa and their closest unicellular and c-AMP-GMP continue to be uncovered. The first usage of relatives, suggesting protist origins for human c-di-NMP sig- cyclic-di-nucleotide (c-di-NMP) signaling in the eukaryote naling. Compared to the limited number of conserved emerged only recently. In dictyostelid social amoebas, domains that detect the second messengers cAMP and cGMP, c-di-GMP is a secreted signal that induces motile amoebas to the domains that detect the c-di-NMPs are surprisingly varied. differentiate into sessile stalk cells. In humans, c-di-NMPs, VC 2013 IUBMB Life, 65(11):897–903, 2013

Keywords: cyclic di-GMP; ; biofilm; cyclic di-AMP; diadenylate cyclase; stalk cell differentiation; Dictyostelium; STING; cGAS; 2030-cGAMP

Introduction karyotes (2–4). As outlined in an excellent recent review (5), c- di-GMP has now far surpassed the prevalence and importance Nucleotide polymers are probably the oldest molecules of life, of cAMP as a second messenger in prokaryotes, while novel as proposed in the RNA-world hypothesis (1). The RNA build- roles still emerge at a rapid rate. In this work, I will compare ing block ATP is the equally ancient carrier of cellular energy, the recently discovered roles of c-di-nucleotides in eukaryotes while its cyclized form, 30,50-cyclic adenosine monophosphate with their established roles in prokaryotes and provide an (cAMP) is a carrier of sensory information in all domains of overview of proteins that mediate synthesis, detection, and life. While the second messenger roles of cAMP and its sister degradation of cyclic di-nucleotides in the two domains. molecule 30,50-cyclic guanosine monophosphate (cGMP) have been studied for over 50 years, the cyclic di-nucleotides 30,50- cyclic diguanylic acid (c-di-GMP), 30,50-cyclic diadenylic acid (c- di-AMP) and 30,50-cyclic adenylic-guanylic acid (c-AMP-GMP) Cyclic-Di-Nucleotide Signalling in were more recently identified as information carriers in pro- Prokaryotes C-di-GMP C-di-GMP was identified in the late 80s as a cofactor that was Additional Supporting Information may be found in the online version of required for activating the cellulose synthase of Gluconoaceto- this article. bacter xylinus (2). However, it took an almost 15 year effort to VC 2013 International Union of Biochemistry and Molecular Biology Volume 65, Number 11, November 2013, Pages 897–903 determine how c-di-GMP was synthesized, detected, and degraded. Cellulose synthase detects c-di-GMP with its associ- *Address correspondence to: Pauline Schaap, MSI=WTB=JBC complex, Dow Street, Dundee DD15EH, United Kingdom. Tel: 144-1382-388078. ated PilZ domain, which is also present in other proteins that Fax: 144-1382-345386. E-mail: [email protected] are directly regulated by c-di-GMP (6,7). The synthetic , Received 16 July 2013; Accepted 28 August 2013 diguanylate cyclase, is related to the type III nucleotidyl DOI 10.1002/iub.1212 transferases, but contains a unique signature GGDEF sequence Published online 17 October 2013 in Wiley Online Library (8–11). The GGDEF domain proteins often harbor an EAL (wileyonlinelibrary.com) domain, which proved to be a 3050-phosphodiesterase (PDE)

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that hydrolyzes one of the phosphodiester bonds of c-di-GMP domains are often degenerate, lacking DGC and PDE activity to form pGpG (8,12). In addition, c-di-GMP can also be con- respectively. In three proteins, PelD, PopA, and CdgG, the verted to GMP by a phosphohydrolase of the HD family that I-site of a defunct GGDEF domain acts as a c-di-GMP receptor, also counts most of the cAMP and cGMP PDEs among its regulating protein function (29–31). In others, such as FimX members. The c-di-GMP PDE is characterized by a signature and LapD, the substrate binding site of a defunct EAL domain HD-GYP sequence motif that differs from the HD motif of the functions as a c-di-GMP receptor (32,33). cAMP and cGMP PDEs (13). This enumeration does not exhaust the variety of c-di-GMP While mechanisms and genes responsible for c-di-GMP targets (Table 1). The biofilm dispersal protein BcdA acts as a metabolism were being identified, the abundance of GGDEF and c-di-GMP sink by binding c-di-GMP (41). c-di-GMP also medi- EAL encoding sequences in emerging bacterial genome sequen- ates signal dependent RNA processing by binding directly to ces suggested that c-di-GMP mediated signaling must be very RNA polynucleotide phosphorylase (PNPase) (35) and c-di-GMP widely used. While this was experimentally confirmed for c-di- regulates gene expression by binding with high affinity to GMP activation of cellulose synthase, cellulose production riboswitches. Riboswitches are sequences in the 50 untrans- appeared to be only one aspect of a suite of c-di-GMP mediated lated regions of bacterial mRNAs that fold into structures responses that cause bacteria to shift from a swarming plank- capable of binding small molecules, which then act to regulate tonic state to a sessile biofilm-associated lifestyle (11,14,15). gene transcription or translation (36,42). The c-di-GMP induced synthesis of cellulose and other exo- This vast, and probably only partially explored, versatility in polysaccharides contributes to the formation of the biofilm c-di-GMP receptors forms a stark contrast with only two intra- matrix, while c-di-GMP induced loss of flagella or other motility cellular receptors for the more ubiquitous signals, cAMP and apparatus, formation of adhesive curli fimbriae or a stalk-like cGMP; that is the cNMP domain (28) and the GAF domain (43). holdfast mark the developmental transition to a sessile life-style Cyclic-di-AMP and Cyclic AMP-GMP (16–18). For pathogenic bacteria, the planktonic state repre- Bacteria use two other c-di-NMPs as second messengers, c-di- sents the virulent acute form of the infection, while the biofilm AMP and c-AMP-GMP, with again unique sets of metabolic state represents the less virulent chronic stage, which is how- and targets. The diadenylate cyclase, DAC or DisA_N, ever more resistant to immune clearance and antibiotics. is part of the DNA scanning protein DisA that monitors Bacillus Other developmental roles for c-di-GMP continue to subtilis DNA for double strand breaks. The presence of Holliday emerge. In the predatory bacterium Bdellovibrio bacteriovorus, junctions, the hallmark of partially repaired breaks, inhibits c- c-di-GMP induces the transition from the axenic mode of feed- di-AMP synthesis by DAC. Further hydrolysis of c-di-AMP to ing to invasion of prey bacteria (19). In the cyanobacterium pApA by YybT=GdpP prevents B. subtilis differentiation into Anabaena sp. c-di-GMP triggers differentiation of nitrogen- spores (3,37). DAC is a novel catalyst with little structural simi- fixing heterocysts (20), while in Streptomyces coelicolor c-di- larity to other mono- or di-nucleotidyl cyclases, and YybT=GdpP GMP regulates the formation of aerial hyphae (21). is a member the DHH domain phosphatases. The DAC domain The targets for c-di-GMP appeared to be numerous and was found to be present in most bacterial phyla and even in are listed in Table 1, which also summarizes the entire reper- Archaea (3). Involvement of c-di-AMP in responses ranging toire of cyclic-di-nucleotide (c-di-NMP) synthetic and hydrolyz- from cell size regulation (44), bacterial cell growth (45), pepti- ing enzymes and targets that are discussed below. As men- doglycan cell wall homeostasis (46) to pathogenicity (47) in a tioned earlier, c-di-GMP binds to the PilZ domain of cellulose range of bacterial species has since been uncovered. The first synthase, but the PilZ domain can also be part of other pro- known target for c-di-AMP is the transcriptional repressor DarR teins, such as the Klebsiella transcription factor MrkH, which (38), again binding c-di-AMP with sequences unrelated to previ- activates the expression of fimbriae or the Pseudomonas aeru- ously known cNMP or c-di-NMP binding domains. ginosa protein Alg44, which is involved in the biosynthesis of The latest addition to the prokaryote c-di-NMPs is c-AMP- the extracellular polysaccharide alginate (22). Additionally, the GMP. This hybrid molecule is synthesized from ATP and GTP by Pilz domain can be expressed on its own, such as PlzA from DncV, a cyclase of Vibrio cholerae, that is unrelated to any other the Lyme disease spirochaete Borrelia burgdorferi, which nucleotidyl cyclases (4). In V. cholerae, c-AMP-GMP promotes mediates c-di-GMP induced motility and infectivity of this intestinal colonization by down-regulating chemotaxis, but the pathogen (23). In addition to the PilZ domain, c-di-GMP has a DncV gene is also present in other proteobacteria and, like c-di- steadily growing number of other targets, such as the tran- AMP and c-di-GMP, may regulate a range of cellular functions. scriptional regulators FleQ (24), Vpst (25), CLP (26), Bcam1349 (27), which each interact with c-di-GMP in a distinctive, but unresolved manner. For CLP and Bcam1349, this may involve Cyclic-Di-Nucleotide Signaling in the intrinsic cNMP domain, the deeply conserved cAMP bind- Eukaryotes ing domain of the bacterial catabolite repressor, CRP, and eukaryote PKA (28). Another c-di-GMP target is the I-site of Dictyostelia some GGDEF domains, which allosterically inhibits c-di-GMP Until 2012, c-di-NMP signaling was considered to be confined synthesis by active DGCs. However, both the GGDEF and EAL to the prokaryote domain. DNA sequences with GGDEF

898 C-di-NMP Signalling in Eukaryotes Functional domains involved in the synthesis, detection and degradation of c-di-NMPs TABLE 1

Organism=c-di-NMP= process Protein name Functional domain Interpro=cd identifier Ref.

Bacteria c-di-GMP

Synthesis GGDEFjClass III diguanylate cyclase (DGC) nucleotidyl cyclase IPR000160jIPR001054 (8) Degradation EAL Diguanylate PDE, EAL domain IPR001633 (8) HD-GYP HD PDEase IPR003607 (13) Detection PilZ PilZ IPR009875 (7) FleQ unknown (24) Clp unknown (26) Bcam1349 unknown (27) FimX EAL substrate binding site IPR001633 (34) PelD RxxD motif in GGDEF domain IPR000160 (29) PNPase unknown (35) Vpst unknown (25) Vc2 Class I Riboswitch GEMM (36) c-di-AMP

Synthesis diadenylate cyclase DisA_N IPR003390 (3) Degradation YybT=GdpP RecJ-like phosphoesterase IPR001667 (37) Detection DarR GATase1_AraC_1 cd03137 (38) c-AMP-GMP

Synthesis DncV (VC0179) no previously identified domain (4) Degradation unknown Detection unknown Dictyostelia c-di-GMP

Synthesis GGDEFjClass III diguanylate cyclase (DgcA) nucleotidyl cyclase IPR000160jIPR001054 (39) Degradation unknown Detection unknown Metazoa 2’3’cGAMP

Synthesis Cyclic GMP-AMP synthase (cGAS) MAB21 (male abnormal 21) IPR024805 (40) Degradation unknown Detection STING STING-C cd12146 (40)

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domains were occasionally detected in the emerging genomes bacterial infections, and this interaction also induces inter- of disparate eukaryotes. However, in almost all cases, the feron gene expression. c-di-GMP binds at a deep cleft between sequences were very similar to prokaryote homologs and the monomers of the constitutively dimeric STING receptor absent from close relatives of the eukaryote in question, indi- (49). cating that they probably originated from contaminating bacte- The question how STING is activated by DNA was recently rial DNA. The exception is a conserved GGDEF domain that is resolved by the finding that human cells synthesize the hybrid present in six genomes, sampled across the full genetic diver- cyclic dinucleotide c-GMP-AMP using the enzyme cGAS, in sity of dictyostelid social amoebas (39). Social amoebas chemo- response to the presence of cytosolic DNA (50,51). This tactically aggregate in response to nutrient stress to form mul- enzyme belongs to the nucleotidyl transferase family, which ticellular aggregates which transform into fruiting structures. includes class III nucleotidyl cyclases, polyadenylate polymer- These structures consist of an aerial mass of spores that is ase, DNA polymerase, and oligoadenylate synthase (OAS1), supported by a column of stalk cells, encased in cellulose. Sev- with the latter being most similar to cGAS. cGAS harbors two eral species, such as D. discoideum have an intermediate N-terminal DNA binding domains, of which the second one is light-sensitive migrating “slug” stage, which in nature serves essential for activation of c-GMP-AMP synthesis by cytosolic to bring the organism to the top layer of the soil for optimal DNA (50). c-GMP-AMP subsequently binds to STING to activate spore dispersal. Dictyostelids extensively use cAMP both in the interferon gene expression. traditional second messenger role and as a secreted first mes- Further studies showed that unlike c-di-AMP and c-di- senger for induction of chemotaxis and spore differentiation GMP, in which both nucleotides are linked from the 30OH of (48). Disruption of the DgcA gene, which encodes the D. discoi- one to the 50-phosphate of the other molecule, cGAS synthe- deum GGDEF domain yielded dgcA-amoebas that form normal sizes a molecule in which the 20OH of GMP is linked to the 50- migrating slugs, but never initiate fruiting body formation. phosphate of AMP, and the 30OH of AMP to the 50-phosphate of DgcA is expressed at the slug tip, where the stalk starts to GMP. This molecule is alternatively named 2030cGAMP (40) or form and was shown to synthesize c-di-GMP. However, unlike c[G(20,50)pA(30,50)p] (52). 2030cGAMP binds to STING with 300- prokaryote c-di-GMP, D. discoideum c-di-GMP does not act as fold higher affinity than c-di-GMP or 3030cGAMP, however, its a second messenger, but is secreted to locally induce stalk cell efficacy for interferon gene induction is only 30 times that of differentiation (39). It is as yet unclear how cells detect and c-di-GMP. Structural studies revealed that 2030cGAMP sits process the c-di-GMP signal, and whether c-di-GMP is hydro- deeper in the cleft between the STING dimers than c-di-GMP, lyzed. No EAL or HD-GYP domains are present in dictyostelid and has three additional polar contacts with STING, explaining genomes and only weak homologies are found to bacterial c- its higher affinity (40). Structural analysis of cGAS showed that di-GMP binding proteins (Chen, Z. and Schaap, P., unpublished it binds double stranded DNA in a non-specific manner by results). As a secreted signal, it may have entirely different electrostatic interactions with the phosphodiester backbone. targets, such as one of the 15 sensor histidine kinases or the These interactions open the catalytic pocket of cGAS and repo- 48 G-protein coupled receptors of D. discoideum. sition several catalytic residues. The formation of a 2050 link- The most intriguing question about c-di-GMP function in age by cGAS is unusual for nucleotidyl cyclases, but similar to dictyostelids is whether it came in through the back door of formation of 2050 oligoadenylates generated by OAS1 (53). lateral gene transfer (LGT), or whether it has deep origins in Detection of foreign DNA by cGAS and subsequent activa- the first eukaryotes. Because a Dictyostelium amoeba con- tion of STING represents a much broader mechanism for the sumes thousands of bacteria every day, LGT is not unlikely. detection of bacterial, viral, and fungal infections than the One piece of evidence is suggestive of deep origins. The D. dis- direct interaction of STING with bacterial c-di-NMPs. It is coideum cellulose synthase, which is essential for stalk forma- therefore likely that the cGAS=STING system evolved inde- tion is more related to bacterial cellulose synthases than to pendently to detect foreign DNA, and that STING activation by other eukaryote (plant) enzymes. Although it does not have a bacterial c-di-NMPs represents fortuitous use of an existing PilZ domain, this domain, and its activation by c-di-GMP, may pathway. have been part of a signaling cassette that early eukaryotes “inherited” from bacteria, with the function of c-di-GMP in stalk formation changing over time. Evolutionary Origins of cGAS and STING To detect co-evolution of cGAS and STING, we screened repre- Humans (and Other Animals) sentative genomes of the major phyla or metazoa and their The detection of viral or bacterial nucleic acids by cellular closest unicellular relative, the choanoflagellate Monosiga brevi- receptors is a critical step in the activation of the human collis, for homologs of the cGAS and STING genes. STING genes innate immune system upon infection. The transmembrane were found in all animal phyla, except Porifera, and also in M. protein STING (stimulator of interferon genes) is an important brevicollis (Fig. 1A). Alignment of the sequences with the struc- link in the pathway that leads from nucleic acid detection to turally characterized human STING, showed that at least 9 out expression of interferon genes. While STING itself does not of 10 residues that are required for binding c-di-NMPs are con- interact with DNA, it was found to bind c-di-GMP derived from served in all phyla, except in the lophotrochozoan Capitella

900 C-di-NMP Signalling in Eukaryotes Phylogenetic analysis of STING and cGAS proteins. The major vertebrate and invertebrate phyla were individually screened for FIG 1 homologs of human STING and cGAS proteins using BlastP. A few representative best hits for each phylum were selected, which were then used for a reverse screen of mammalian genomes. For STING, the reverse search yielded mouse and bat STING proteins. However, invertebrate cGAS homologs yielded mammalian Mab21 proteins as bidirectional hits. Protein sequences were aligned using M-coffee (54). After deletion of segments with poor consensus alignment, sequences were sub- jected to Bayesian inference for establishment of phylogenetic relationships between proteins (55). Analyses were run for 1 million generations under a mixed amino-acid model with rate variation between sites estimated by a gamma distribution. Bayesian inference posterior probabilities (BIPPs) of tree nodes are indicated by colored dots. Gene identifiers of the proteins are annotated with functional domain architectures and color-coded to represent the phyla from which they are derived. Corre- sponding species names are listed in the legends to Supporting Information Figures 1 and 2. telata (Supporting Information Fig. S1), which also has an aber- Concluding Remarks rant functional domain architecture (Fig. 1A). Similar to cAMP signaling, the rapidly expanding field of c-di- The cGAS catalytic and DNA binding sequences are con- NMP signaling owes its existence to the original painstaking tained within the PFAM Mab21 domain, which was first identification of a small biologically active molecule (2). detected in proteins involved in embryonic development. Whereas molecular genetics and genomics are currently the Blastp search with human cGAS detects Mab21 proteins most powerful tools to unravel gene function, they have limited throughout metazoa, but arthropod=lophotrochozoan Mab21 ability to predict the repertoire of small molecules that many proteins are much more related to mammalian sensu stricto of the cognate enzymes might synthesize. Our current under- Mab21 proteins than to cGAS (Fig. 1B). Sequence alignment standing of particularly developmental signaling is therefore (Supporting Information Fig. S2) shows that the vertebrate biased toward peptide signals that were discovered through cGAS homologs share all residues required for DNA and sub- genetics, leaving potentially important non-peptide mediated strate binding with human cGAS, while the invertebrate pro- signaling undiscovered. The diverse functions of monomeric teins lack most of the DNA binding residues and several of the and polymeric nucleotides in information storage, energy substrate binding residues. Interestingly, conservation of transfer, and signaling depend on the making and breaking of essential residues in the chordates Branchiostoma floridae and phosphodiester bonds. All life forms have a very large reper- Saccoglossus kowalevski is intermediate between cGAS and toire of enzymes to perform these functions and their potential Mab21, with the proteins of the hemichordate S. kowalevski for producing biologically active molecules may as yet be and the cephalochordate B. floridae being somewhat more vastly underestimated. The expertise of dedicated biochemists, similar to Mab21 and cGAS, respectively (Supporting Informa- rather than geneticists, will however be needed to identify tion Figs. S2 and 1B). This probably marks the emergence of these molecules. cGAS in the chordate=vertebrate lineage. Contrary to expectation, the comparative analysis shows that cGAS and STING did not evolve together and that metazoa References and their protist ancestors detected c-di-NMPs long before 0 0 [1] Gilbert, W. (1986) Origin of life—the RNA world. Nature 319, 618. they could synthesize 2 3 cGAMP. The function of this early [2] Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger-Ohana, P., et al. metazoan c-di-NMP detection system presents an intriguing (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic avenue for further study. diguanylic acid. Nature 325, 279–281.

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