Effector-mediated membrane disruption controls cell death in CBASS antiphage defense

Duncan-Lowey, Brianna; McNamara-Bordewick, Nora K.; Tal, Nitzan; Sorek, Rotem; Kranzusch, Philip J.

doi:10.1016/j.molcel.2021.10.020

Cited by 63 publications

(39 citation statements)

References 46 publications

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“… 11 ): cUA; Y. aleksiciae CdnE (ref. 36 ): 3′3′-cUU; Drosophila eugracilis cGLR1 (ref. 31 ): 3′2′-cGAMP; Mus musculus cGAS (ref.…”

Section: Methodsmentioning

confidence: 99%

“…CBASS effector function was measured in E. coli using conditions that result in autoactivation of V. cholerae DncV 3′3′-cGAMP synthesis as previously described 36 . E. coli BL21(DE3) competent cells (NEB) were transformed with three plasmids encoding V. cholerae CapV (pBAD33), the CBASS effector B. pseudomallei Cap5 (pET16), and either WT or catalytically inactive (H44A/H113A) T4 Acb1 (pTU175) 51 .…”

Section: Methodsmentioning

confidence: 99%

“… 23 ), Y. aleksiciae CdnE (ref. 36 ) cloned into a pBAD vector, and E. coli PycC (ref. 2 ) cloned under its native promoter into the plasmid pSG1.…”

Section: Methodsmentioning

confidence: 99%

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Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Hobbs

Wein

et al. 2022

Nature

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The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation1,2. Phages use several strategies to defeat host CRISPR and restriction-modification systems3–10, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3′3′-cyclic GMP–AMP define a mechanism of metal-independent hydrolysis 3′ of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.

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“… 11 ): cUA; Y. aleksiciae CdnE (ref. 36 ): 3′3′-cUU; Drosophila eugracilis cGLR1 (ref. 31 ): 3′2′-cGAMP; Mus musculus cGAS (ref.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Hobbs

Wein

et al. 2022

Nature

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“…These enzymes, called c GAS/ D ncV-like n ucleotidyltransfer ases (CD-NTases) produce cyclic oligonucleotides, such as cGAMP, during bacteriophage (phage) infection. When assayed in heterologous expression systems, cyclic oligonucleotides activate a downstream effector (Cohen et al, 2019; Lau et al, 2020; Ye et al, 2020; Lowey et al, 2020; Duncan-Lowey et al, 2021), which is proposed to kill the cell and prevent phage replication (Lopatina et al, 2020). This strategy of bacterial immunity was coined c yclic-oligonucleotide- b ased a nti-phage s ignaling s ystems (CBASS) (Cohen et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Bacteriophages antagonize cGAS-like immunity in bacteria

Huiting

Athukoralage

Guan

et al. 2022

Preprint

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The recently discovered cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is related to eukaryotic cGAS-STING anti-viral immunity and is present in diverse prokaryotes. However, our understanding of how CBASS detects, inhibits, and co-evolves with phages is limited because CBASS function has only been studied in reconstituted heterologous systems. Here, we identify a phage-encoded CBASS antagonist (acbIIA1, anti-cbass type II-A gene 1) necessary for phage replication in the presence of endogenous CBASS immunity in Pseudomonas aeruginosa. acbIIA1 homologs are encoded by numerous lytic and temperate phages infecting Gram-negative bacteria. Deletion of acbIIA1 renders multiple phages susceptible to CBASS, but phages can then escape immune function via mutations in the major capsid gene. These mutants suggest that CBASS is activated by, or targets, the late-expressed phage capsid. Together, we establish a native model system to study CBASS and identify a common phage-encoded CBASS antagonist, demonstrating that CBASS is a bona fide anti-phage immune system in nature.

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“…Type I CBASS systems encode only a cGAS enzyme and an effector protein, while the majority of CBASS systems (Types II, III, and IV) also encode additional proteins with proposed roles in phage sensing and cGAS activation. The mechanisms underlying CBASS-induced cell death have recently been the focus of numerous studies 4,10,12 , but the role of the additional proteins remains largely unknown.…”

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confidence: 99%

cGASylation by a bacterial E1-E2 fusion protein primes antiviral immune signaling

Ledvina

et al. 2022

Preprint

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In all organisms, innate immune pathways sense viral infection and rapidly activate potent immune responses while maintaining a high degree of specificity to prevent inappropriate activation (autoimmunity). In humans, the innate-immune receptor cGAS detects viral infection to produce the nucleotide second messenger cGAMP, which initiates STING-dependent antiviral signaling. Bacteria encode predecessors of the cGAS-STING pathway, termed cyclic oliogonucleotide-based antiphage signaling systems (CBASS), and bacterial cGAS detects bacteriophage infection to produce cGAMP. How bacterial cGAS activation is controlled, however, remains unknown. Here, we show that the CBASS-associated protein Cap2 primes bacterial cGAS for activation through a ubiquitin transferase-like mechanism. A cryoelectron microscopy structure of the Cap2-cGAS complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling the eukaryotic E1 protein ATG7 and the E2 proteins ATG10 and ATG3. The structure captures a reactive-intermediate state with the cGAS C-terminus extending into the Cap2 E1 active site and conjugated to AMP. We find that Cap2 ligates the cGAS C-terminus to a target molecule in cells, a process we call cGASylation. cGASylation primes cGAS for a ~50-fold increase in cGAMP production. We further demonstrate that Cap2 activity is balanced by a specific endopeptidase, Cap3, which deconjugates cGAS and antagonizes antiviral signaling. Our data demonstrate that bacteria control immune signaling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of E1 and E2 machinery across the kingdoms of life.

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Effector-mediated membrane disruption controls cell death in CBASS antiphage defense

Cited by 63 publications

References 46 publications

Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity

Bacteriophages antagonize cGAS-like immunity in bacteria

cGASylation by a bacterial E1-E2 fusion protein primes antiviral immune signaling

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