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

Abstract

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.

An expanding arsenal of immune systems that protect bacteria from phages

“…Intriguingly, type II CBASS anti-phage defense systems also contain ancillary genes ( cap2 and cap3 ), which encode enzymes with E1, E2, and JAB domains (Millman et al, 2020b). These enzymes were recently shown to conjugate the cGAS-like defense protein of CBASS onto unknown targets in the bacterial cell to boost immunity (Ledvina et al, 2022). It is possible that the parallel enzymes in the ISG15-like system similarly function to conjugate the ISG15-like protein onto cellular targets as part of the immune function.…”

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage

“…We also identified operons encoding proteins related to eukaryotic ubiquitin signaling machinery, including so-called 6a systems that encode a large protein with E2-like, E1-like, and JAB protease-like domains (Burroughs et al , 2009; Iyer et al , 2006). Notably, this protein shares strong homology to the Cap2 protein in Type II CBASS systems (also classified as 6b systems), which conjugates the C-terminus of its cognate CD-NTase to an unknown target to regulate anti-phage signaling (Ledvina et al , 2022). We also identified CapH and CapP-like proteins associated with 6e systems, which encode a protein predicted to possess multiple ubiquitin-like β-grasp domains and an E1-like protein ( Figure 6F ).…”

“…While so-called Type I CBASS systems encode only a CD-NTase and a cell-killing effector protein, the majority of CBASS systems encode ancillary genes putatively involved in infection sensing and/or CD-NTase activation (Millman et al , 2020; Burroughs et al , 2015). Type II CBASS systems encode two proteins, Cap2 and Cap3, that are related to eukaryotic ubiquitination machinery and are required for protection against phage (Cohen et al , 2019; Ledvina et al , 2022). Type III CBASS systems, meanwhile, encode peptide-binding HORMA domain proteins (Cap7 and Cap8) that are proposed to bind specific peptides to sense infection and then activate their associated CD-NTase (Ye et al , 2020).…”

Structure and activity of a bacterial defense‐associated 3′‐5′ exonuclease

“…Many CBASS systems encode proteins related to important eukaryotic signaling machinery: for example, a set of CBASS systems encoding STING‐like effectors are the likely evolutionary predecessors of the mammalian cGAS‐STING DNA‐sensing innate immune pathway 8 . Some CBASS systems encode regulatory proteins related to eukaryotic HORMA domain and TRIP13 signaling proteins, 3,7 while others encode regulators with homology to eukaryotic ubiquitin signaling machinery 3,9 …”

“…8 Some CBASS systems encode regulatory proteins related to eukaryotic HORMA domain and TRIP13 signaling proteins, 3,7 while others encode regulators with homology to eukaryotic ubiquitin signaling machinery. 3,9 CBASS systems are remarkably diverse, and many systems encode putative effectors, regulators, and other ancillary proteins whose biochemical functions and biological roles remain unknown. Around 250 of the 6,233 identified CBASS systems have been reported to encode a predicted 3 0 -5 0 exonuclease with homology to DEDDhfamily DNA/RNA exonucleases.…”