Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

Jenson, Justin M.; Li, Tuo; Du, Fenghe; Ea, Chee-Kwee; Chen, Zhijian J.

doi:10.1038/s41586-023-05862-7

Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

Journal Article · 27 February 2023 · Nature (London)

DOI:https://doi.org/10.1038/s41586-023-05862-7· OSTI ID:2423723

; ; Du, Fenghe; Ea, Chee-Kwee;

Abstract

cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection^1–3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP–AMP (cGAMP)^4,5, which leads to the expression of antimicrobial genes^6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered^8–11. These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% containCap2andCap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively^8,12. Although these proteins are required to prevent infection of some bacteriophages⁸, the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC02-06CH11357

OSTI ID:: 2423723

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7956 Vol. 616; ISSN 0028-0836

Publisher:: Nature Publishing Group

Country of Publication:: United States

Language:: English

References (37)

Calculations and Publication-Quality Illustrations for Analytical Ultracentrifugation Data Brautigam, Chad A. Methods in Enzymology https://doi.org/10.1016/bs.mie.2015.05.001	book	January 2015
Coordinated Regulation of Accessory Genetic Elements Produces Cyclic Di-Nucleotides for V. cholerae Virulence Davies, Bryan W.; Bogard, Ryan W.; Young, Travis S. Cell, Vol. 149, Issue 2 https://doi.org/10.1016/j.cell.2012.01.053	journal	April 2012
Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling Burroughs, A. Maxwell; Zhang, Dapeng; Schäffer, Daniel E. Nucleic Acids Research, Vol. 43, Issue 22 https://doi.org/10.1093/nar/gkv1267	journal	November 2015
Bacterial origins of human cell-autonomous innate immune mechanisms Wein, Tanita; Sorek, Rotem Nature Reviews Immunology, Vol. 22, Issue 10 https://doi.org/10.1038/s41577-022-00705-4	journal	April 2022
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega Sievers, Fabian; Wilm, Andreas; Dineen, David Molecular Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1038/msb.2011.75	journal	January 2011
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
Phaser crystallographic software McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D. Journal of Applied Crystallography, Vol. 40, Issue 4 https://doi.org/10.1107/S0021889807021206	journal	July 2007
Bacterial cGAS-like enzymes synthesize diverse nucleotide signals Whiteley, Aaron T.; Eaglesham, James B.; de Oliveira Mann, Carina C. Nature, Vol. 567, Issue 7747 https://doi.org/10.1038/s41586-019-0953-5	journal	February 2019
Evolutionary Origins of cGAS-STING Signaling Margolis, Shally R.; Wilson, Stephen C.; Vance, Russell E. Trends in Immunology, Vol. 38, Issue 10 https://doi.org/10.1016/j.it.2017.03.004	journal	October 2017
Inference of Macromolecular Assemblies from Crystalline State Krissinel, Evgeny; Henrick, Kim Journal of Molecular Biology, Vol. 372, Issue 3 https://doi.org/10.1016/j.jmb.2007.05.022	journal	September 2007
Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation Letunic, Ivica; Bork, Peer Nucleic Acids Research, Vol. 49, Issue W1 https://doi.org/10.1093/nar/gkab301	journal	April 2021
CBASS phage defense and evolution of antiviral nucleotide signaling Duncan-Lowey, Brianna; Kranzusch, Philip J. Current Opinion in Immunology, Vol. 74 https://doi.org/10.1016/j.coi.2022.01.002	journal	February 2022
cGAS in action: Expanding roles in immunity and inflammation Ablasser, Andrea; Chen, Zhijian J. Science, Vol. 363, Issue 6431 https://doi.org/10.1126/science.aat8657	journal	March 2019
Optimization of T4 phage engineering via CRISPR/Cas9 Duong, Michelle M.; Carmody, Caitlin M.; Ma, Qinqin Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-75426-6	journal	October 2020
Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae Severin, Geoffrey B.; Ramliden, Miriam S.; Hawver, Lisa A. Proceedings of the National Academy of Sciences, Vol. 115, Issue 26 https://doi.org/10.1073/pnas.1801233115	journal	June 2018
A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core Zimmermann, Lukas; Stephens, Andrew; Nam, Seung-Zin Journal of Molecular Biology, Vol. 430, Issue 15 https://doi.org/10.1016/j.jmb.2017.12.007	journal	July 2018
RNA-guided editing of bacterial genomes using CRISPR-Cas systems Jiang, Wenyan; Bikard, David; Cox, David Nature Biotechnology, Vol. 31, Issue 3, p. 233-239 https://doi.org/10.1038/nbt.2508	journal	January 2013
A ubiquitin-like system mediates protein lipidation Ichimura, Yoshinobu; Kirisako, Takayoshi; Takao, Toshifumi Nature, Vol. 408, Issue 6811 https://doi.org/10.1038/35044114	journal	November 2000
Structure of the Escherichia coli ThiS−ThiF Complex, a Key Component of the Sulfur Transfer System in Thiamin Biosynthesis ^† ^, ^‡ Lehmann, Christopher; Begley, Tadhg P.; Ealick, Steven E. Biochemistry, Vol. 45, Issue 1 https://doi.org/10.1021/bi051502y	journal	January 2006
An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol Liu, Huanting; Naismith, James H. BMC Biotechnology, Vol. 8, Issue 1 https://doi.org/10.1186/1472-6750-8-91	journal	January 2008
Bacteriophages inhibit and evade cGAS-like immune function in bacteria Huiting, Erin; Cao, Xueli; Ren, Jie Cell, Vol. 186, Issue 4 https://doi.org/10.1016/j.cell.2022.12.041	journal	February 2023
STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling Ishikawa, Hiroki; Barber, Glen N. Nature, Vol. 455, Issue 7213 https://doi.org/10.1038/nature07317	journal	August 2008
CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing Labun, Kornel; Montague, Tessa G.; Krause, Maximilian Nucleic Acids Research, Vol. 47, Issue W1 https://doi.org/10.1093/nar/gkz365	journal	May 2019
High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis Keller, Sandro; Vargas, Carolyn; Zhao, Huaying Analytical Chemistry, Vol. 84, Issue 11 https://doi.org/10.1021/ac3007522	journal	June 2012
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway Sun, L.; Wu, J.; Du, F. Science, Vol. 339, Issue 6121 https://doi.org/10.1126/science.1232458	journal	December 2012
Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling Schuck, Peter Biophysical Journal, Vol. 78, Issue 3 https://doi.org/10.1016/S0006-3495(00)76713-0	journal	March 2000
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Cyclic GMP-AMP Is an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA Wu, J.; Sun, L.; Chen, X. Science, Vol. 339, Issue 6121 https://doi.org/10.1126/science.1229963	journal	December 2012
WebLogo: A Sequence Logo Generator Crooks, Gavin E.; Hon, Gary; Chandonia, John-Marc Genome Research, Vol. 14, Issue 6, p. 1188-1190 https://doi.org/10.1101/gr.849004	journal	May 2004
Cyclic GMP–AMP signalling protects bacteria against viral infection Cohen, Daniel; Melamed, Sarah; Millman, Adi Nature, Vol. 574, Issue 7780 https://doi.org/10.1038/s41586-019-1605-5	journal	September 2019
Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems Millman, Adi; Melamed, Sarah; Amitai, Gil Nature Microbiology https://doi.org/10.1038/s41564-020-0777-y	journal	August 2020
Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling Houtman, Jon C. D.; Brown, Patrick H.; Bowden, Brent Protein Science, Vol. 16, Issue 1 https://doi.org/10.1110/ps.062558507	journal	January 2007
Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection Otten, Elsje G.; Werner, Emma; Crespillo-Casado, Ana Nature, Vol. 594, Issue 7861 https://doi.org/10.1038/s41586-021-03566-4	journal	May 2021
PHROG: families of prokaryotic virus proteins clustered using remote homology Terzian, Paul; Olo Ndela, Eric; Galiez, Clovis NAR Genomics and Bioinformatics, Vol. 3, Issue 3 https://doi.org/10.1093/nargab/lqab067	journal	June 2021

Similar Records

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling

Journal Article · 2019 · Nature (London) · OSTI ID:1497105

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression

Journal Article · 2019 · Nature Communications · OSTI ID:1524667

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila

Journal Article · 2021 · Nature (London) · OSTI ID:1820446

Related Subjects

Science & Technology - Other Topics

Ubiquitin-like conjugation by bacterial cGAS enhances anti-phage defence

Citation Formats

References (37)

Similar Records

Related Subjects