Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 3'3'3'-cyclic AMP-AMP-AMP and 3'3'3'-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense.

CRISPR-Cas immune systems provide bacteria with adaptive immunity against bacteriophages, but they are often transcriptionally repressed to mitigate auto-immunity. In some cases, CRISPR-Cas expression increases in response to a phage infection, but the mechanisms of induction are largely unknown, and it is unclear whether induction occurs strongly and quickly enough to benefit the bacterial host. In S. pyogenes, Cas9 is both an immune effector and auto-repressor of CRISPR-Cas expression. Here, we show that phage-encoded anti-CRISPR proteins relieve Cas9 auto-repression and trigger a rapid increase in CRISPR-Cas levels during a single phage infective cycle. As a result, fewer cells succumb to lysis, leading to a striking survival benefit after multiple rounds of infection. CRISPR-Cas induction also reduces lysogeny, thereby limiting a route for horizontal gene transfer. Altogether, we show that Cas9 is not only a CRISPR-Cas effector and repressor but also a phage sensor that can mount an anti-anti-CRISPR transcriptional response.