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Bacterial cGAS-like enzymes synthesize diverse nucleotide signals

Nature volume 567, pages 194–199 (2019)Cite this article

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Abstract

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

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Fig. 1: Bacteria synthesize cyclic UMP–AMP.
Fig. 2: Conserved active-site residues dictate CD-NTase specificity.
Fig. 3: Immune detection of a pyrimidine-containing CDN.
Fig. 4: CD-NTases synthesize diverse nucleotide products and form a family of enzymes abundant in many bacterial phyla.
Fig. 5: Bacterial synthesis and host recognition of a cyclic trinucleotide.

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Data availability

All data supporting the findings of this study are available within the Article and associated Supplementary Information. X-ray crystallographic coordinates and structure factor files are available from the PDB: RmCdnE apo (6E0K); RmCdnE–Apcpp–Upnpp (6E0L); EmCdnE apo (6E0M); EmCdnE–GTP–Apcpp (6E0N); EmCdnE–pppA[3′–5′]pA (6E0O); RECON–cAAG (6M7K). CD-NTase sequences and information on CD-NTase-encoding bacteria are available in Supplementary Table 2 and CD-NTase effector genes sequences are available in Supplementary Table 3. CD-NTase alignments for tree construction are provided as Source Data for Fig. 4a and Source Data are also available for Extended Data Fig. 8b in the online version of the paper. Source gel images are available in Supplementary Fig. 1.

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Acknowledgements

We thank K. McCarty and V. Cabrera for technical assistance; S. Wilson for advice; M. Reniere for reading the manuscript; T. Levin for input on CD-NTase tree construction; T. Wyche and M. Henke from the HMS ICCB Longwood Screening Facility and the staff at the HMS East Quad NMR Facility for their advice and technical support; A. Govande and W. Zhou for providing purified proteins; C. Miller for assistance with X-ray crystallography data collection; and the members of the Mekalanos and Kranzusch laboratories for advice and discussions. This work was funded by the Claudia Adams Barr Program for Innovative Cancer Research (P.J.K.), Richard and Susan Smith Family Foundation (P.J.K.), Charles H. Hood Foundation (P.J.K.), a Cancer Research Institute CLIP Grant (P.J.K.), NIH/NIAID R01AI018045 and R01AI026289 (J.J.M.), the Searle Scholars Program (A.S.Y.L.) and a Sloan Research Fellowship (A.S.Y.L.). A.T.W. is supported as a fellow of The Jane Coffin Childs Memorial Fund for Medical Research, C.C.d.O.M. is supported as a Cancer Research Institute/Eugene V. Weissman Fellow and B.R.M. is supported by the NIH T32 Cancer Immunology training grant (5T32CA207021-02). X-ray data were collected at the Lawrence Berkeley National Laboratory Advanced Light Source beamlines 8.2.1 and 8.2.2, this work used Northeastern Collaborative Access Team beamlines 24-ID-C and 24-ID-E (P30 GM124165), a Pilatus detector (S10RR029205), an Eiger detector (S10OD021527) and Argonne National Laboratory Advanced Photon Source (DE-AC02-06CH11357).

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Nature thanks Regine Hengge, Joshua Woodward and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. Olga Danilchanka

    Present address: Merck Research Laboratories, Merck & Co. Inc., Kenilworth, NJ, USA

Authors and Affiliations

  1. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Aaron T. Whiteley, James B. Eaglesham, Carina C. de Oliveira Mann, Benjamin R. Morehouse, Brianna Lowey, Olga Danilchanka, John J. Mekalanos & Philip J. Kranzusch

  2. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Aaron T. Whiteley, James B. Eaglesham, Carina C. de Oliveira Mann, Benjamin R. Morehouse, Brianna Lowey, Eric A. Nieminen & Philip J. Kranzusch

  3. HHMI Mass Spectrometry Laboratory, University of California, Berkeley, Berkeley, CA, USA

    David S. King

  4. Department of Biology, Brandeis University, Waltham, MA, USA

    Amy S. Y. Lee

  5. Parker Institute for Cancer Immunotherapy at Dana-Farber Cancer Institute, Boston, MA, USA

    Philip J. Kranzusch

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Contributions

Experiments were designed and conceived by A.T.W., J.J.M. and P.J.K. Gene identification, activity screening and bacterial assays were performed by A.T.W. Biochemical experiments were performed by A.T.W., J.B.E. and P.J.K. Structural experiments and analysis were performed by J.B.E. and P.J.K. Nucleotide synthesis and purification was performed by A.T.W., B.L. and P.J.K. Mass spectrometry was performed by C.C.d.O.M. and D.S.K. NMR was performed by B.R.M. Cloning assistance was provided by E.A.N. Observations and strains were contributed by O.D. Bioinformatics and cell assays were performed by A.S.Y.L. Figures were prepared by A.T.W., J.B.E. and P.J.K. The manuscript was written by A.T.W., J.B.E., J.J.M. and P.J.K. All authors contributed to editing the manuscript and support the conclusions.

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Correspondence to John J. Mekalanos or Philip J. Kranzusch.

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Competing interests

Harvard Medical School and the Dana-Farber Cancer Institute have patents pending for CD-NTase technologies on which A.T.W., J.B.E., J.J.M. and P.J.K are listed as inventors.

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Extended data figures and tables

Extended Data Fig. 1 Detailed characterization of CdnE, a cUMP–AMP synthase.

a, b, Titration of reaction buffer pH in steps of 0.2 pH units. Recombinant DncV or CdnE was incubated with α32P-radiolabelled ATP, CTP, GTP and UTP at varying pH and the reactions were treated with alkaline phosphatase and visualized by PEI–cellulose TLC. CdnE activity is optimal at a pH of approximately 9.4, and this reaction condition was used in further experiments. Data are representative of two independent experiments. c, PEI–cellulose TLC of products after incubation of the indicated enzyme, wild-type CdnE or active site mutant CdnE with α32P-radiolabelled ATP, CTP, GTP and UTP as in Fig. 1b. Mutations that ablate the CdnE Mg2+-coordinating, active-site residues eliminate all detectable activity. Data are representative of three independent experiments. d, Nuclease P1 sensitivity of CDN products. Nuclease P1 specifically cleaves 3′–5′, canonical phosphodiester bonds. DncV and CdnE products are completely digested in the presence of P1 and alkaline phosphatase, whereas only one bond of 2′,3′-cGAMP is susceptible to digestion, producing the linear G[2′–5′]pA product. Data are representative of three independent experiments. e, Workflow of nucleotide production for mass spectrometry and NMR analysis. f, Anion-exchange chromatography of a CdnE reaction with ATP and UTP, eluted with a gradient of buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated before pooling for further analysis. g, Anion-exchange chromatography (IEX) fractions from f were separated by silica TLC, visualized by ultraviolet-light shadowing and compared to a radiolabelled reaction to confirm the appropriate absorbance at 254 nm (A254) peak. Fractions were pooled and concentrated before mass spectrometry and NMR analysis. h, k, l, 3′,3′-Cyclic uridine monophosphate–adenosine monophosphate proton-NMR spectrum (h) and associated magnified spectra (k, l) 1H NMR (400 MHz): δΗ 8.40 (s, 1H), 8.17 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 6.15 (s, 1H), 5.75 (s, 1H), 5.55 (d, J = 8.2 Hz, 1H), 5.00–4.90 (m, 2H), 4.80 (d, J = 4.5 Hz, 1H) 4.70–4.61 (m, 1H), 4.55–4.38 (m, 4H), 4.17–4.02 (m, 2H). i, j, 3′,3′-Cyclic uridine monophosphate–adenosine monophosphate phosphate-NMR spectrum (i) and associated magnified spectrum (j). 31P{1H} NMR (162 MHz): δP −1.59 (s, 1P), −1.65 (s, 1P).

Extended Data Fig. 2 Order of the DncV, cGAS and CdnE reactions.

a–c, Incubation of CD-NTase enzymes with non-hydrolysable nucleotides traps reaction intermediates and identifies the order of the reaction. Left, PEI–cellulose TLC analysis of reactions as in Fig. 1b, in which individual NTPs have been replaced by their non-hydrolysable nucleotide analogues. Exposed γ-phosphates are labelled in parentheses to indicate that these are removed by phosphatase treatment before analysis. Data are representative of three independent experiments. Right, previously determined reaction mechanisms (DncV and cGAS)10,51 and proposed reaction mechanism for CdnE.

Extended Data Fig. 3 Detailed analysis of RmCdnE.

a, A thermophilic homologue of CdnE from R. marinus (RmCdnE) also synthesizes cUMP–AMP. Recombinant proteins were incubated with α32P-radiolabelled NTPs as indicated at either 37 °C (CdnE) or 70 °C (RmCdnE) and the reactions were visualized by PEI–cellulose TLC as in Fig. 1b. Data are representative of two independent experiments. b, Active site of RmCdnE superimposed with structures of cGAS (6CTA30) and DncV (4TY010). c, The analogous position to Asn166 was mutated in CdnE to a serine and that protein, CdnEN166S, was characterized further. Reactions, which were analysed as in Fig. 1b, demonstrate that CdnEN166S loses pyrimidine specificity. Data are representative of two independent experiments. d, Structure-based sequence alignment of CD-NTases, annotated with secondary structure features of RmCdnE and human cGAS (6CTA30). Mg2+-coordinating active-site residues are highlighted in red; blue arrows, cyan/pink annotations and purple highlights represent secondary structure; and the analogous residues to RmCdnE Asn166 are highlighted in orange.

Extended Data Fig. 4 Detailed structural analysis of EmCdnE.

a, Sequence alignment of CdnE homologues in Fig. 2c, annotated with RmCdnE secondary structure features. Mg2+-coordinating active-site residues are highlighted in red and the analogous residues to RmCdnE Asn166 are highlighted in orange. Yersinia enterocolitica (WP_050915017); P. aeruginosa (WP_096075289); Xanthomonas arboricola (WP_104644370); Xenorhabdus nematophila (WP_010848498); Bordetella parapertussis (WP_015040391); Burkholderia cepacia complex (WP_006482377); R. marinus (RmCdnE, WP_014072508); L. pneumophila (WP_042646516); Mycobacterium avium (WP_062886322); E. meningoseptica (EmCdnE, WP_016200549); Staphylococcus aureus (WP_031901603); Enterococcus faecalis (WP_050492554); Bacteroides thetaiotaomicron (WP_062695386). b, Biochemical deconvolution of EmCdnE, which has a natural serine substitution at the Asn166 analogous site. Recombinant protein was incubated with NTPs as indicated and reactions were visualized as in Fig. 1b. Data are representative of three independent experiments. c, Reactions of EmCdnE incubated with α32P-radiolabelled NTPs and non-hydrolysable nucleotide analogues as indicated and visualized as in Fig. 1b. Data are representative of three independent experiments. d, Anion exchange chromatography of an EmCdnE reaction with ATP and GTP, eluted with a gradient of buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated before pooling for further analysis. e, Anion exchange chromatography fractions from d were separated by silica TLC, visualized by ultraviolet-light shadowing and compared to a radiolabelled reaction to confirm the appropriate peak. Fractions were pooled and concentrated before mass spectrometry analysis. Mass spectrometry confirmed synthesis of products with masses corresponding to c-di-AMP, cGAMP and c-di-GMP. f, Crystal structure of EmCdnE in complex with GTP and non-hydrolysable ATP capturing the ‘first state’ structure before NTP hydrolysis. Mg2+ ions are omitted for clarity. g, Magnified cut-away of the active site of the complex shown in f, confirming the position of a serine at the analogous site to RmCdnE Asn166. Nucleotide and metal 2Fo − Fc electron densities are contoured at 1σ. h, Magnified cut-away of the active site of the EmCdnE–pppApA structure, capturing the ‘second state’ after the first reaction has occurred to form a linear intermediate, but before CDN formation. Nucleotide and metal 2Fo – Fc electron densities are contoured at 1σ. g, h, Mg2+ ions are shown in green. i, Biochemical deconvolution of mutant EmCdnE reverted to the ancestral asparagine at the Asn166 analogous site. This mutant loses preference for producing cyclic dipurine molecules and instead produces more pyrimidine-containing CDN products. Reactions were visualized as in Fig. 1b. Data are representative of two independent experiments.

Extended Data Fig. 5 cUMP–AMP recognition helps to define innate immune receptor specificity.

a, e, Quantification of nucleotide interactions with the host receptors STING (a) or RECON (e), measured using a radiolabelled nucleotide bound to a concentration gradient of host protein, separated in a native PAGE gel shift (0, 4, 20, 100 μM protein). Quantification of the gels shown in b and f. Data are representative of n = 2 independent experiments. b, f, Native PAGE gel shift analysis of STING (b) or RECON (f) complex formation with indicated radiolabelled CDNs. Proteins are titrated at 0 (−), 4, 20 and 100 μM. STING readily binds all cyclic dipurine species, but does not form a high-affinity complex with cUMP–AMP. RECON readily binds all 3′,3′-CDN species that contain at least one adenine base, including cUMP–AMP. Data are representative of two independent experiments. c, In-cell STING reporter assay. Induction of an IFNβ reporter in HEK293T cells transfected with a concentration gradient of plasmid-overexpressing enzymes as indicated. DncV and CdnE were expressed with N-terminal MBP tags and IFNβ reporter induction was compared as fold over empty vector, shown as (−). Data are mean ± s.e.m. for n = 3 technical replicates and are representative of two independent experiments. d, Western blot of MBP-tagged DncV and CdnE expressed from plasmids analysed in c to validate in vivo expression. Data are representative of two independent experiments. Gel source data are available in Supplementary Fig. 1. g, Gel shift analysis as in f, with protein titration to measure the relative affinity of the RECON–cUMP–AMP interaction. Protein concentrations listed below. Data are representative of 2 independent experiments.

Extended Data Fig. 6 A biochemical screen of CD-NTases from diverse bacterial genera.

a, Chart of the number of bacterial genomes (n = 16,717) that have CD-NTases from clusters in Fig. 4a. See also Supplementary Table 2. b, Taxa of genome-sequenced bacteria isolated with unique CD-NTase genes, phyla are indicated in bold; Proteobacteria and Firmicutes are further divided by order and visualized by shades of colour. c–f, Type CD-NTases interrogated for product synthesis. Purified proteins were incubated with α32P-radiolabelled NTPs under different reaction conditions (indicated pH and divalent cation) and reaction products were visualized by either PEI–cellulose or silica TLC as in Fig. 1b and Fig. 4c, respectively. g, CD-NTase expression level and purity. Coomassie-stained SDS–PAGE analysis of purified CD-NTase enzymes used in each reaction. Data are representative of two independent experiments.

Extended Data Fig. 7 Detailed biochemical analysis of LpCdnE02.

a, Biochemical deconvolution of LpCdnE02 (CD-NTase057), analysed as in Fig. 1c, demonstrates specific synthesis of cyclic dipyrimidine products. Data are representative of three independent experiments. b, Nuclease sensitivity of the LpCdnE02 product, as described in Extended Data Fig. 1d. Data are representative of three independent experiments. c, Incubation of LpCdnE02 with non-hydrolysable nucleotides, as described in Extended Data Fig. 2. Non-hydrolysable UTP completely blocks the reaction, indicating the first step requires attack of the αP from UTP. However, the product formed when non-hydrolysable CTP is present cannot be distinguished from c-di-UMP in this assay, and it is unclear whether the reaction proceeds through a pppCpU reaction intermediate. Data are representative of three independent experiments. d, Anion exchange chromatography of a LpCdnE02 reaction with UTP and CTP, eluted with a gradient of buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated before pooling for further analysis. e, Anion exchange chromatography fractions from d were separated by silica TLC, visualized by ultraviolet-light shadowing and compared to a radiolabelled reaction to confirm the appropriate peak. Fractions were pooled and concentrated before mass spectrometry analysis. f, Mass spectrometry confirmed synthesis of c-di-UMP as the major product of LpCdnE02. g, Mass spectrometry confirmed synthesis of cCMP–UMP as a minor product of LpCdnE02.

Extended Data Fig. 8 CD-NTases are encoded in conserved, poorly understood operons on mobile genetic elements.

a, Operon structure for CD-NTases selected for in-depth characterization showing the conserved protein domains in CD-NTase adjacent genes (see Fig. 4a–c). Conserved operons were identified previously and operons are vertically organized by similarity to one another22. Where found, linked genes demonstrating that CD-NTases are encoded on mobile genetic elements are indicated. b, CD-NTases and their adjacently encoded ‘effector’ proteins were coexpressed in E. coli and bacterial colony formation was quantified by spot-dilution analysis. CD-NTases were inducibly expressed from a chloramphenicol-resistance (CmR) vector and effectors were inducibly expressed from a carbenicillin-resistance (CarbR) vector. Bacteria that express cognate CD-NTase–effector plasmids or control plasmids were plated on medium containing both inducers and incubated for 24 h at 37 °C. Data were not determined (N.D.) for CD-NTase036, because the effector was toxic to E. coli under non-inducing conditions. Data are the mean ± s.e.m. of three independent experiments. c, Spot-dilution analysis of bacteria expressing the indicated cognate CD-NTase–effector pairs, quantified as in b. The CD-NTase036–effector pair was not analysed in this assay. Colony morphology indicates a potential interaction for some combinations; however, it is unclear how specific or meaningful this may be. Data are representative of three independent experiments.

Source data

Extended Data Fig. 9 Detailed biochemical analysis of EcCdnD02.

a, Titration of reaction buffer pH in steps of 0.2 pH units. Recombinant EcCdnD02 was incubated with α32P-radiolabelled NTPs at varying pH and the reactions were analysed and visualized by PEI–cellulose or silica TLC as in Fig. 1b and Fig. 4c, respectively. Silica TLC identified two products, denoted the major (blue triangle) and minor (red triangle) product. Quantification of TLC spots is shown at the bottom. Data are representative of two independent experiments. b, Biochemical deconvolution of EcCdnD02, recombinant protein was incubated with NTPs as indicated and analysed by TLC as in a. Data are representative of three independent experiments. c, Nuclease digestion of the EcCdnD02 product. Conventional nuclease digestion includes addition of a phosphatase, in this experiment reactions were first treated with Antarctic phosphatase to remove remaining NTPs and were then heat-inactivated. Next, reactions were untreated, treated with nuclease P1 (specific for 3′–5′-phosphodiester bonds) only or treated with nuclease P1 and phosphatase to remove exposed phosphate groups. 3′,3′-cGAMP (DncV) and EcCdnD02 product are digested into AMP and GMP constituents, which are phosphatase-sensitive. cAMP (CyaA) is insensitive to P1 digestion and cyclic monophosphates are phosphatase-resistant. These data demonstrate that the EcCdnD02 product does not contain a cyclic monophosphate. Data are representative of three independent experiments. d, Incubation of EcCdnD02 with non-hydrolysable nucleotides, as described in Extended Data Fig. 2. Non-hydrolysable ATP completely blocks the reaction, indicating the first step requires attack of the αP from ATP. However, when non-hydrolysable GTP is present the possible intermediates (pp(c)pGpA, pp(c)pGpApA, or pppApA) cannot be distinguished in this assay and it is unclear how the reaction proceeds. Silica TLC is not suited for analysing non-hydrolysable nucleotides, because they do not migrate beyond the origin. Data are representative of three independent experiments. e, Anion exchange chromatography of a EcCdnD02 reaction with ATP and GTP, eluted with a gradient of buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated before pooling for further analysis. f, g, 3′,3′,3′-Tricyclic adenosine monophosphate–adenosine monophosphate–guanosine monophosphate phosphate-NMR spectrum (f) and associated magnified spectrum (g). 31P{1H} NMR (162 MHz): δP −0.65 (s, 1P), −0.70 (s, 1P), −0.75 (s, 1P). h–j, 3′,3′,3′-Tricyclic adenosine monophosphate–adenosine monophosphate–guanosine monophosphate proton-NMR spectrum (h) and associated magnified spectra (i, j). 1H NMR (400 MHz): δΗ 8.43 (s, 1H), 8.39 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 6.15 (d, J = 7.0 Hz, 1H), 6.12 (d, J = 7.0 Hz, 1H), 5.92 (d, J = 7.5 Hz, 1H), 5.00–4.78 (m, 6H), 4.69–4.58 (m, 3H), 4.3–4.2 (m, 6H).

Extended Data Fig. 10 Structural analysis of cAAG inhibition of RECON.

a, cAAG interactions with STING or RECON, radiolabelled nucleotides incubated with a concentration gradient of each protein, separated in a native PAGE gel shift (0, 4, 20, 100 μM protein). Data are representative of two independent experiments. b, Co-crystal structure of the RECON–cAAG complex shown as cartoon (left) and surface (right). c, Overlay and orientation of RECON ligands cAAG, c-di-AMP (PDB 5UXF), co-substrate NAD (PDB 3LN3) demonstrate three individual binding pockets. d, Schematic representation of residues from RECON that interact with cAAG. Green dotted lines indicate hydrogen bonds, grey dotted lines indicate hydrophobic interactions. e, Magnified cutaways of individual RECON binding pockets as in d. f, Mammalian innate-immune sensors recognize CD-NTase products with overlapping specificities. 2′,3′-cGAMP and c-di-GMP are detected by STING; 3′,3′-cGAMP and c-di-AMP are detected by both STING and RECON; and cUMP–AMP and cAAG are detected by RECON.

Supplementary information

Supplementary Information

This file contains a detailed discussion of how to identify CD-NTases and bacteria that encode a given CD-NTase of interest. It also includes Supplementary Table 1, a summary of data collection, phasing and refinement statistics, and Supplementary Figure 1, which includes original gel source data.

Reporting Summary

Supplementary Table 2

CD-NTases and CD-NTase encoding bacteria. Sequences of CD-NTases selected for screening and subsequent in-depth analysis, CD-NTases identified in bacteria, and bacteria predicted to encode a CD-NTase.

Supplementary Table 3

CD-NTase effector genes. Sequences of CD-NTase effector genes encoded adjacently to CD-NTases chosen for in-depth analysis.

Supplementary Data

CD-NTase alignment and tree. Alignment and tree of CD-NTase amino acid sequences from Supplementary Table 2. These provide the source data for Fig. 4a.

Source data

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Whiteley, A.T., Eaglesham, J.B., de Oliveira Mann, C.C. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019). https://doi.org/10.1038/s41586-019-0953-5

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